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 / Ampicillin-Stammlösung, 5 ml 100x -Mercaptoethanol Stammlösung und 10 ng / ml bFGF. Halten sterilfiltriert Medium bei 4 ° C für nicht mehr als 10 Tage. </li><li> Bereiten mTeSR1 Serum-freien Medium durch die Kombination der folgenden: 400 ml mTeSR1 Grundmedium, 100 ml 5x zu ergänzen, und 5 ml 100x Penicillin / Ampicillin-Stammlösung. Halten Medium bei 4 ° C für nicht mehr als 10 Tage. </li><li> Bereiten 10x Kollagenase IV-Stammlösung: 0,5 g abwiegen Kollagenase IV Macht, undlösen sie mit 50 ml DMEM / F12 und Filter zu sterilisieren. Aliquots machen und versehen Sie sie bei -20 ° C für Monate. </li><li> Bereiten 0,1% Gelatine-Lösung: 0,5 g abwiegen Gelatine (aus Rinderhaut, Typ B) Kraft und löst ihn mit VE-Wasser. Halten Autoklaven sterilisierte Lösung bei Raumtemperatur für Wochen. </li><li> Bereiten KSR Differenzierungsmedium durch die Kombination der folgenden: 410 ml DMEM, 75 ml KSR, 5 ml 100x nicht-essentielle Aminosäure-Stammlösung, 5 ml 100x Penicillin / Ampicillin-Stammlösung, 5 ml 100x -Mercaptoethanol Stammlösung. Halten sterilfiltriert Medium bei 4 ° C für nicht mehr als 10 Tage. HINWEIS: KSR variiert von Charge zu Charge, die die Differenzierung Effizienz beeinträchtigen können. Es ist daher besser, mehrere Chargen von KSR zu testen, um das beste für die Differenzierung zu finden. </li><li> Bereiten N2 Differenzierungsmedium durch die Kombination der folgenden: 98 ml DMEM, 1 ml 100x N2 Ergänzung und 1 ml 100x Penicillin / Ampicillin-Stammlösung. Halten sterilfiltriert Medium bei 4° C für nicht mehr als 10 Tage. </li><li> Bereiten B27 Differenzierungsmedium durch die Kombination der folgenden: 480 ml Neurobasalmedium, 10 ml 50x B27 ergänzen, 5 ml 100x Glutamax Stammlösung und 5 ml 100x Penicillin / Ampicillin-Stammlösung. Halten sterilfiltriert Medium bei 4 ° C für nicht mehr als 10 Tage. </li></ol> 2. Kultur von hES-Zellen und hiPSCs auf MEF Feeder-Zellen Die H9 hES wurden von WiCell Research Institute und hiPSCs wurden in der Reijo Pera Labor durch Retrovirus-vermittelte Transduktion von Yamanaka gegründet Faktoren OCT3 / 4, SOX2, Klf4 und c-MYC 18. <ol><li> Mindestens einen Tag vor der Zelldurchgang, bereiten einige Gelatineplatten durch Zugabe von 1 ml 0,1% Gelatine auf 1 und der 6-Well-Platten, Platten und Inkubation bei 37 ° C für mindestens 30 min. </li><li> Nach der Inkubation aspirieren Gelatine von den Platten und Samen Bestrahlung inaktiviert MEF-Zellen auf die Platten in einer Dichte von 1,6 x 10 5 Zellen/ Well in MEF Medium mit einer Zählkammer, um die Zellen zu zählen. HINWEIS: Optional MEF Feeder schon nach 3 Tage vor der Zelldurchgang vorzubereiten. </li><li> Passage-Zellen einen Tag nach Plattierung MEF Anleger: absaugen Medium von hPSCs, fügen Sie 1 mg / ml Kollagenase IV-Zellen in 1 ml / Vertiefung und Inkubation Zellen bei 37 ° C für 1 Stunde. </li><li> Während dieser Inkubation, Waschen MEF Feeder einmal mit PBS, und fügen Sie dann warm (37 ° C) serumhaltigem HPSC Kulturmedium bei 1,5 ml / well. </li><li> Nach 1 Stunde, tippen Sie auf der Kulturplatte ein paar Mal, so dass die meisten der undifferenzierte Kolonien von den Platten lösen, während die differenzierte Kolonien befestigt bleiben. Sorgfältig sammeln die schwimmende Kolonien, und sie in 15-ml-Tube. </li><li> Die Platte vorsichtig waschen einmal mit warmem (37 ° C) DMEM / F12 und übertragen DMEM / F12 in den gleichen 15-ml-Tube. </li><li> Zentrifugieren Sie die Röhrchen bei 179 g für 3 min. </li><li> Saugen Sie das Medium aus den Röhren, man aufpassen, dass das Pellet nicht zu stören. Krieg hinzufügenm HPSC Kulturmedium, Pipette die Zellen nach oben und unten mehrere Male, um sie in kleinen Gruppen zu brechen und mischen sie gut. </li><li> Übertragen Sie die Zellen auf neue MEF Feeder bei 1: 3-1: 6-Verhältnis. </li><li> Ändern Sie das Medium jeden Tag und Durchgang die Zellen alle 5-6 Tage. </li></ol> 3. Herstellung von Zellen zur Differenzierung <ol><li> Mindestens einen Tag vor der Herstellung der Zellen, bereiten einige Matrigel Platten (typischerweise 3 Wells einer 6-Well-Platte). Verdünnen Sie die Matrigel mit kaltem (4 ° C) DMEM / F12 um 1:40 und 1 ml pro Vertiefung Matrigel 6-Well-Platten. Inkubieren Matrigel Platten bei 4 ° C über Nacht. Hinweis: Man kann Matrigel Platten mit Parafilm versiegeln haben und lagern bei 4 ° C solange eine Woche. </li><li> Verwenden Zellen (siehe Schritt 2) 4 Tage nach Durchgang. Entfernen Sie alle differenzierte Kolonien von den Platten. Absaugen Medium von der Kulturplatte, 1 ml pro Vertiefung Accutase, und Inkubation Zellen bei 37 ° C für 5 min. </li><li> Während der Inkubation bereiten einige GelAtins Platten durch Zugabe von 1 ml Gelatine 1 und der 6-Well-Platten. Legen Sie diese Platten und die Matrigel-Platten einen Tag vor im Inkubator bereit. </li><li> Nach der 5-minütigen Inkubation oder wenn die Zellen haben sich halbschwimmend, Pipetten Zellen nach oben und unten mehrere Male, um sie in Einzelzellen zu machen. </li><li> Sammeln Sie einzelne Zellen in 15-ml-Röhrchen und zentrifugieren bei 258 g für 3 min. </li><li> Die Zellen mit einer geeigneten Menge an Serum, HPSC Kulturmedium, und fügen Thiazovivin bis zu einer Endkonzentration von 2 uM. </li><li> Übertragung von Zellen auf Gelatine-beschichteten Platten in einer 1: 1-Verhältnis, und inkubieren Zellen bei 37 ° C für 30 min bis MEF Feeder entfernen. </li><li> Übertragen Sie die Zellen in ein 15 ml konischen Röhrchen, fügen Sie entsprechende Serum mit HPSC Medium und zählen Sie die Zellen mit einer Zählkammer. </li><li> Zentrifugieren Sie die Zellen bei 258 g für 3 min. </li><li> Während Zentrifuge, fügen Thiazovivin zu mTeSR1 geeignete Medium, um eine Konzentration von 2 um zu machen. </li><li> Nach Zentrifuge, einspirate das Medium mit der geeigneten mTeSR1 Medium mit Thiazovivin so, dass es 1,8 x 10 5 Zellen / ml Medium. </li><li> Nehmen Sie die Matrigel Platten aus dem Inkubator, saugen Sie den Matrigel und 2 ml der gemischten Zellen auf 1 und der 6-Well-Platten. HINWEIS: Die Zelldichte sollte 3,6 x 10 4 Zellen / cm 2 Oberfläche ist. </li><li> Bringen Sie die Platten wieder in den Inkubator, und lassen die Zellen legen für 24 Stunden. </li><li> 24 Stunden nach dem Ausstreichen der Zellen absaugen alte Medium und 2 ml Medium pro neue mTeSR1 gut und lassen Zellen wachsen weitere 24 Stunden vor Beginn der Differenzierung. </li></ol> Zelldifferenzierung <ol><li> Starten Differenzierung 48 Stunden nach der Neubelegung der Zellen auf Matrigel Platten. </li><li> Absaugen Kulturmedium von der Platte, waschen Sie die Zellen einmal mit PBS, und 2 ml der folgenden Differenzierungsmedium pro Well: KSR Differenzierungsmedium mit 10 uM SB431542 und 100 nM LDN-193 ergänzt189. Mark diesen Tag als D0 der Differenzierung. </li><li> Ändern Medium täglich von D0 bis D20. HINWEIS: Man kann Medium zur Verwendung nicht mehr als 2 Tage zu einer Zeit vorzubereiten. </li><li> Verwenden Sie das folgende Medium für D1 und D2: KSR Differenzierungsmedium mit 10 uM SB431542, 100 nM LDN-193189, 0,25 um SAG 2 um purmorphamine und 50 ng / ml FGF8b ergänzt. </li><li> Verwenden Sie das folgende Medium für D3 und D4: KSR Differenzierungsmedium mit 10 uM SB431542, 100 nM LDN-193189, 0,25 um SAG 2 um purmorphamine, 50 ng / ml FGF8b und 3 um CHIR99021 ergänzt. </li><li> Verwenden Sie das folgende Medium für D5 und D6: 75% KSR Differenzierungsmedium plus 25% N2 Differenzierungsmedium mit 100 nM LDN-193189, 0,25 um SAG 2 um purmorphamine, 50 ng / ml FGF8b und 3 um CHIR99021 ergänzt. </li><li> Verwenden Sie das folgende Medium für D7 und D8: 50% KSR Differenzierungsmedium plus 50% N2 Differenzierungsmedium mit 100 nM LDN-193189 und 3 um CHIR99021 ergänzt.</li><li> Verwenden Sie das folgende Medium für D9 und D10: 25% KSR Differenzierungsmedium plus 75% N2 Differenzierungsmedium mit 100 nM LDN-193189 und 3 um CHIR99021 ergänzt. </li><li> Verwenden Sie das folgende Medium für D11 und D12: B27 Differenzierungsmedium mit 3 um CHIR99021, 10 ng / ml BDNF, 10 ng / ml GDNF, 1 ng / ml TGF3, 0,2 mM Ascorbinsäure und 0,1 mM cAMP ergänzt. </li><li> Verwenden Sie das folgende Medium für den Rest der Differenzierung: B27 Differenzierungsmedium mit 10 ng / ml BDNF, 10 ng / ml GDNF, 1 ng / ml TGF3, 0,2 mM Ascorbinsäure und 0,1 mM cAMP ergänzt. </li><li> Bei etwa D16 der Differenzierung, bereiten einige Poly-L-Ornithin / Laminin / Fibronektin-Platten. Verdünnen Poly-L-Ornithin-Stammlösung mit kaltem (4 ° C) PBS bis 15 pg / ml, 1 ml auf 1 und der 6-Well-Platten, die Platten und Inkubation bei 37 ° C über Nacht. </li><li> Saugen Sie den Poly-L-Ornithin-Lösung, waschen Sie die Platten 3 mal mit sterilem Wasser und dann an der Luft trocknen die Platten. </li><li> Verdünnen Laminin Lager Solution mit kaltem (4 ° C) PBS auf 1 pg / ml, 1 ml einer gut 6-Well-Platten, die Platten und Inkubation bei 37 ° C über Nacht. </li><li> Saugen Sie den Laminin-Lösung, waschen Sie die Platten 3 mal mit sterilem Wasser und dann an der Luft trocknen die Platten. </li><li> Verdünnen Fibronektin-Stammlösung mit kaltem (4 ° C) PBS auf 2 pg / ml, 1 ml auf 1 und der 6-Well-Platten, die Platten und Inkubation bei 37 ° C über Nacht. </li><li> Dichtungsplatten mit Parafilm und sie bei 4 ° C für bis zu zwei Wochen. </li><li> Nach 20 Tagen der Differenzierung, Ausstrich der Zellen an die Poly-L-Ornithin / Laminin / Fibronektin-Platten. Absaugen Differenzierungsmedium, 1 ml warmem (37 ° C) bis 1 Accutase auch der 6-Well-Platten und Inkubation Zellen bei 37 ° C für 5 min. </li><li> Während der Inkubation, legen Sie die Poly-L-Ornithin / Laminin / Fibronektin-Platten in den Inkubator. </li><li> Nach der 5-minütigen Inkubation oder wenn die Zellen haben sich halbschwimmende, sanft Pipette Zellen nach oben und unten mehrere Male, ummachen sie in einzelne Zellen. </li><li> Sammeln Sie die einzelnen Zellen in 15 ml konischen Röhrchen, und fügen Sie die entsprechende Menge an Neurobasalmedium zur Zellzählung, die je nach Ausdehnung variieren (nach 11 Tagen der Differenzierung, Zellen 15-20 fach erweitern). Zählen Sie die Zellen mit einer Zählkammer. </li><li> Zentrifugieren Sie die Zellen bei 258 g für 3 min. Überstand wird abgesaugt, und resuspendieren Zellen mit B27 Differenzierungsmedium, so daß die Zellkonzentration 1 x 10 6 Zellen / ml Medium. </li><li> Absaugen Fibronektin von den Platten, zweimal waschen mit PBS, und 2 ml Zellen 1 gut 6-Well-Platten. HINWEIS: Die Zelldichte für Replattierung sollte 2 x 10 5 Zellen / cm 2 Oberfläche ist. </li><li> Ändern Medium 24 Stunden später, um alle losen toten Zellen zu entfernen. </li><li> Ändern Medium jeden zweiten Tag, bis die gewünschten Zeitpunkte für einen bestimmten Experiment. </li></ol>

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The authors declare that they have no competing financial interests.

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">
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			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
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			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
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			<td height="21" style="height:21px;width:308px;">cAMP</td>
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			<td style="width:119px;">D0627</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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</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

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.

Regie dopaminergen Neuron Differenzierung von humanen pluripotenten Stammzellen

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

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.3K Aufrufe.  Stanford University School of Medicine. 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’s disease.

Serie: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with 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 (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

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 ...

Forschung

Neurowissenschaften

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

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

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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 ...

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

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

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

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

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

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

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

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

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 ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Organotypische Kulturen des adulten humanen Kortex als Ex-vivo-Modell für die Transplantation und Validierung humaner Stammzellen

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...