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 Préparation de milieux de culture <ol><li> Préparer la souris fibroblastes embryonnaires (MEF) moyen en combinant la suivante: 445 ml de DMEM, 50 ml de sérum de veau fœtal (FBS), et 5 ml 100x pénicilline / ampicilline solution stock. Maintenir le filtre milieu stérilisé à 4 ° C pendant au plus 14 jours. </li><li> Préparer le milieu de culture HPSC en combinant les éléments suivants contenant du sérum: 385 ml de DMEM / F12, 100 ml de remplacement de sérum de finale (KSR), 5 ml 100x non essentiels solution solution acide aminé d&#39;achat d&#39;actions, 5 ml 100x pénicilline / ampicilline actions, 5 ml 100x mercaptoéthanol solution mère, et 10 ng / ml de bFGF. Maintenir le filtre milieu stérilisé à 4 ° C pendant au plus 10 jours. </li><li> Préparer un milieu sans sérum mTeSR1 en combinant les éléments suivants: 400 ml mTeSR1 milieu de base, 100 ml 5x supplément, et 5 ml 100x pénicilline / ampicilline solution stock. Gardez milieu à 4 ° C pendant pas plus de 10 jours. </li><li> Préparer IV solution mère 10x collagénase: peser 0,5 g puissance collagénase IV, etdissoudre avec 50 ml de DMEM / F12 et stériliser par filtration. Faire aliquotes et les stocker à -20 ° C pendant plusieurs mois. </li><li> Préparer une solution de gélatine à 0,1%: peser 0,5 g de gélatine (peau de bovin, type B) de puissance et dissoudre avec de l&#39;eau désionisée. Gardez solution stérilisée en autoclave à la température ambiante pendant des semaines. </li><li> Préparer milieu de différenciation KSR en combinant la suivante: 410 ml de DMEM, 75 ml KSR, 5 ml 100x solution non acide aminé essentiel actions, 5 ml 100x pénicilline / ampicilline solution stock, 5 ml 100X mercaptoéthanol solution de stock. Maintenir le filtre milieu stérilisé à 4 ° C pendant au plus 10 jours. REMARQUE: KSR varie d&#39;un lot à l&#39;, ce qui peut affecter l&#39;efficacité de la différenciation. Il est donc préférable de tester plusieurs lots de KSR pour trouver la meilleure solution pour la différenciation. </li><li> Préparer milieu de différenciation N2 en combinant les éléments suivants: 98 ml de DMEM, 1 ml 100x supplément N2 et 1 ml 100x pénicilline / ampicilline solution stock. Maintenir le filtre milieu stérilisé à 4° C pendant au plus 10 jours. </li><li> Préparer milieu de différenciation de B27 en combinant la suivante: 480 ml de milieu neurobasal, 10 ml 50x B27 supplément, 5 ml 100X Glutamax solution mère, et 5 ml 100x pénicilline / ampicilline solution stock. Maintenir le filtre milieu stérilisé à 4 ° C pendant au plus 10 jours. </li></ol> 2. culture des CSEh et hiPSCs sur des cellules nourricières MEF Les CSEh H9 proviennent de l&#39;Institut de recherche et WiCell hiPSCs ont été établis dans le laboratoire Reijo Pera par transduction rétrovirale médiation de Yamanaka facteurs OCT3 / 4, SOX2, KLF4 et c-MYC 18. <ol><li> Au moins un jour avant le passage des cellules, la préparation des plaques de gélatine par addition de 1 ml de gélatine à 0,1% à 1 puits de plaques à 6 puits et incuber les plaques à 37 ° C pendant au moins 30 min. </li><li> Après incubation, les plaques de gélatine aspiration, et l&#39;irradiation des semences cellules MEF inactivées sur les plaques à la densité de 1,6 x 10 5 cellules/ Puits dans le milieu MEF utilisant un hémocytomètre pour compter les cellules. REMARQUE: préparer option mangeoires MEF dès 3 jours avant le passage de la cellule. </li><li> cellules Passage d&#39;un jour après l&#39;étalement mangeoires MEF: Aspirer le milieu de hPSCs, ajouter 1 mg / ml de collagénase IV de cellules à 1 ml / puits et incuber les cellules à 37 ° C pendant 1 heure. </li><li> Pendant cette incubation, laver les mangeoires MEF fois avec du PBS, puis ajoutez chaud (37 ° C) HPSC milieu de culture contenant du sérum à 1,5 ml / puits. </li><li> Après 1 heure, appuyez sur la plaque de culture une couple de fois de sorte que la plupart des colonies indifférenciées se détacher des plaques, tandis que les colonies différenciées restent attachés. Recueillir soigneusement les colonies flottantes, et de les transférer en tube de 15 ml. </li><li> Lavez délicatement la plaque une fois avec chaud (37 ° C) DMEM / F12 et transférer DMEM / F12 dans le même tube de 15 ml. </li><li> Centrifuger les tubes à 179 g pendant 3 min. </li><li> Aspirer le milieu des tubes, en faisant attention de ne pas perturber le culot. Ajouter guerremilieu de culture m HPSC, la pipette les cellules de haut en bas plusieurs fois pour les briser en petits groupes et mélangez-les bien. </li><li> Transférer les cellules sur de nouveaux départs MEF à 1: 3-1: 6 rapport. </li><li> Changer le support chaque jour et le passage des cellules tous les 5-6 jours. </li></ol> 3 Préparation des cellules pour la différenciation <ol><li> Au moins un jour avant la préparation de cellules, préparer des plaques de Matrigel (typiquement 3 puits d&#39;une plaque à 6 puits). Diluer avec du Matrigel froid (4 ° C) du milieu DMEM / F12 à 01:40 et ajouter 1 ml de Matrigel par puits de plaques à 6 puits. Incuber les plaques de Matrigel à 4 ° C pendant une nuit. NOTE: On peut sceller les plaques de Matrigel avec du Parafilm et les stocker à 4 ° C pendant aussi longtemps qu&#39;une semaine. </li><li> Utiliser des cellules (voir étape 2) 4 jours après le passage. Retirez toutes les colonies différenciées des plaques. Aspirer le milieu de la plaque de culture, ajouter 1 ml Accutase par puits, et incuber les cellules à 37 ° C pendant 5 min. </li><li> Pendant l&#39;incubation, préparer quelques gelatin plaques par addition de 1 ml de gélatine à 1 puits de plaques à 6 puits. Placez ces plaques et les Matrigel plaques préparées la veille dans l&#39;incubateur. </li><li> Après la 5 minutes d&#39;incubation ou lorsque les cellules sont devenues semi-flottant, les cellules de pipette de haut en bas plusieurs fois pour en faire des cellules individuelles. </li><li> Recueillir des cellules individuelles dans tubes de 15 ml et centrifuger à 258 g pendant 3 min. </li><li> Resuspendre les cellules avec suffisamment de sérum contenant du milieu de culture, et HPSC Thiazovivin ajouter à la concentration finale de 2 uM. </li><li> Transfert des cellules sur des plaques recouvertes de gélatine à 1: 1, et les cellules incuber à 37 ° C pendant 30 min pour éliminer les mangeoires MEF. </li><li> Transférer les cellules dans un tube conique de 15 ml, ajouter un milieu contenant du sérum approprié HPSC et compter les cellules avec un hémocytomètre. </li><li> Centrifuger les cellules à 258 g pendant 3 min. </li><li> Au cours de centrifugeuse, ajouter Thiazovivin de s&#39;approprier moyen mTeSR1 pour obtenir une concentration de 2 uM. </li><li> Après centrifugation, unespirate le milieu et ajouter mTeSR1 milieu approprié avec Thiazovivin sorte qu&#39;il n&#39;y a 1,8 x 10 5 cellules / ml de milieu. </li><li> Sortez les plaques Matrigel de l&#39;incubateur, aspirer le Matrigel et ajouter 2 ml de cellules mixtes sur 1 puits des plaques de 6 puits. REMARQUE: La densité cellulaire doit être de 3,6 x 10 4 cellules / cm 2 de surface. </li><li> Remettre les plaques de retour à l&#39;incubateur, et que les cellules se fixent pendant 24 heures. </li><li> 24 heures après l&#39;étalement des cellules, aspirer ancien milieu et ajouter 2 ml nouveau média mTeSR1 par puits et laisser les cellules se développent pour un autre 24 heures avant le début de la différenciation. </li></ol> La différenciation des cellules <ol><li> Lancer la différenciation 48 heures après les plaquant les cellules sur des plaques de Matrigel. </li><li> Aspirer le milieu de culture de la plaque, se laver les cellules une fois avec du PBS, et ajouter 2 ml de milieu de différenciation suivante par puits: milieu de différenciation KSR complété avec 10 uM SB431542 et 100 nM LDN-193189. Marquer ce jour D0 de différenciation. </li><li> Changer le milieu tous les jours de D0 à D20. NOTE: On peut préparer le milieu pour une utilisation pas plus de 2 jours à la fois. </li><li> Utilisez le milieu suivant pour D1 et D2: milieu de différenciation KSR complété avec 10 uM SB431542, 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, et 50 ng / ml FGF8b. </li><li> Utilisez le milieu suivant pour D3 et D4: milieu de différenciation KSR complété avec 10 uM SB431542, 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, 50 ng / ml FGF8b, et 3uM CHIR99021. </li><li> Utilisation du support suivant pour D5 et D6: 75% de milieu de différenciation KSR plus 25% milieu de différenciation N2 complété avec 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, 50 ng / ml FGF8b, et 3 uM CHIR99021. </li><li> Utilisez le milieu suivant pour D7 et D8: 50% de milieu de différenciation KSR plus 50% de milieu de différenciation N2 complété avec 100 nM LDN-193189 et 3 uM CHIR99021.</li><li> Utilisez le milieu suivant pour D9 et D10: 25% KSR milieu de différenciation plus 75% de milieu de différenciation N2 complété avec 100 nM LDN-193189 et 3 uM CHIR99021. </li><li> Utilisation du support suivant pour D11 et D12: milieu de différenciation B27 additionné de 3 uM CHIR99021, 10 ng / ml de BDNF, 10 ng / ml GDNF 1 ng / ml TGF3, 0,2 mM d&#39;acide ascorbique et 0,1 mM d&#39;AMPc. </li><li> Utilisation du support suivant pour le reste de la différenciation: milieu de différenciation B27 supplémenté avec 10 ng / ml de BDNF, 10 ng / ml GDNF 1 ng / ml TGF3, l&#39;acide ascorbique 0,2 mM et 0,1 mM d&#39;AMPc. </li><li> A peu près à D16 de la différenciation, de préparer des poly-L-ornithine laminine / plaques / fibronectine. Diluer poly-L-ornithine solution stock de froid (4 ° C) PBS à 15 pg / ml, ajouter 1 ml à 1 puits de plaques 6 puits et incuber les plaques à 37 ° C pendant la nuit. </li><li> Aspirer la solution poly-L-ornithine, laver les plaques 3 fois avec de l&#39;eau stérile, puis sécher à l&#39;air les plaques. </li><li> Diluer la laminine stocks solution de froid (4 ° C) PBS à 1 pg / ml, ajouter 1 ml d&#39;un puits de plaques 6 puits et incuber les plaques à 37 ° C pendant la nuit. </li><li> Aspirer la solution de laminine, laver les plaques 3 fois avec de l&#39;eau stérile, puis sécher à l&#39;air les plaques. </li><li> Diluer la solution de fibronectine du stock de froid (4 ° C) PBS à 2 pg / ml, ajouter 1 ml à 1 puits de plaques 6 puits et incuber les plaques à 37 ° C pendant la nuit. </li><li> plaques d&#39;étanchéité avec du parafilm et placer à 4 ° C pendant jusqu&#39;à deux semaines. </li><li> Après 20 jours de différenciation, Réensemencement cellules aux plaques / laminine / fibronectine Poly-L-ornithine. milieu de différenciation Aspirer, ajouter 1 ml chaude (37 ° C) Accutase à 1 puits de plaques 6 puits et incuber les cellules à 37 ° C pendant 5 min. </li><li> Au cours de l&#39;incubation, placer les poly-L-ornithine laminine / plaques / fibronectine dans l&#39;incubateur. </li><li> Après la 5 minutes d&#39;incubation ou lorsque les cellules sont devenues semi-flottant, en douceur les cellules de pipette de haut en bas plusieurs fois àrendre dans des cellules individuelles. </li><li> Recueillir les cellules individuelles dans 15 ml tubes coniques, et ajouter la quantité appropriée de milieu neurobasal pour le comptage des cellules, qui varie en fonction de l&#39;expansion (après 11 jours de différenciation, les cellules peuvent augmenter 15-20 fois). Compter les cellules en utilisant un hématimètre. </li><li> Centrifuger les cellules à 258 g pendant 3 min. Aspirer le surnageant et remettre en suspension les cellules avec du milieu de différenciation B27 de sorte que la concentration cellulaire est de 1 x 10 6 cellules / ml de milieu. </li><li> Aspirer la fibronectine à partir des plaques, laver deux fois avec du PBS, et ajouter 2 ml de cellules à une puits de plaques à 6 puits. REMARQUE: La densité cellulaire pour réensemencement doit être de 2 x 10 5 cellules / cm 2 de surface. </li><li> Changer le milieu de 24 heures plus tard pour enlever les cellules mortes seules. </li><li> Changer le milieu tous les deux jours jusqu&#39;à ce que les points de temps souhaités pour une expérience donnée. </li></ol>

<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>

The authors declare that they have no competing financial interests.

Les cellules souches pluripotentes humaines (y compris les cellules hES hiPSCs) et peuvent être différenciées in vitro afin de générer la plupart, sinon tous, les types de notre corps, y compris les neurones dopaminergiques, qui a été démontré dans des études précédentes 19 cellules. Ici, basée sur la connaissance de dopaminergique développement embryonnaire des neurones 20 et protocoles publiés par les autres laboratoires 14,15, nous avons optimisé les condition...

Dopaminergique (DA) des neurones peuvent être trouvés dans plusieurs régions du cerveau, y compris le mésencéphale, hypothalamus, la rétine et bulbes olfactifs. Les neurones DA A9 dans la substantia nigra pars compacta (SNpc) et un comportement de commande de mouvement en projetant dans le striatum du cerveau antérieur et formant le système moteur extrapyramidal. La dégénérescence des neurones DA A9 conduit à la maladie de Parkinson (PD), qui est la deuxième maladie neurodégénérative humaine la plus courante et actuellement incurable. la thérapie de remplacement cellulaire est l&#39;une des stratégies les plus prometteuses pour le traitement de PD 1, par conséquent, il ya eu un grand intérêt pour dériver neurones A9 DA à partir de cellules souches pluripotentes humaines (hPSCs), y compris les cellules souches embryonnaires humaines (CSEh) et la cellules dernières anthropiques souches pluripotentes (hiPSCs). Beaucoup de recherches ont tenté de tirer les neurones A9 DA de hPSCs en utilisant diverses méthodes. Les premiers rapports tous les neurones DA générés par un neuronalstade rosette progénitrices. En co-culture avec MS5 2 ou PA6 3-5 cellules stromales avec ou sans facteurs de croissance externe pour 2-4 semaines, L. Studer et collègues et trois autres groupes induits succès CSEh pour produire des rosettes de neurones. Ils ont ensuite enrichi ces rosettes par dissection mécanique ou digestion enzymatique pour une différenciation plus poussée. Dans d&#39;autres rapports, les chercheurs ont généré des rosettes de neurones par corps embryoïdes (EB) la différenciation de culture 6-9 flottants. Par la suite, les chercheurs ont établi un procédé de différenciation de la monocouche à base de 10,11, où ils étalées cellules hES et hiPSCs sur la matrice extra-cellulaire, ajouter différents facteurs de croissance dans la culture pour induire la différenciation de hPSCs de neurones dopaminergiques, qui imite l&#39;vivo DA embryonnaire du développement dans des neurones . Bien que toutes ces études obtenus tyrosine hydroxylase (TH) cellules exprimant certaines caractéristiques des neurones dopaminergiques, le processus de différenciation ensemble est temps et de travail consommant,généralement inefficaces, et plus important encore, l&#39;identité A9 de ces neurones n&#39;ont pas été démontrée dans la plupart des études, sauf celle avec Lmx1a expression ectopique 12. Récemment, une nouvelle plaque de plancher (FP) du protocole à base a été développé 13-16, dans lequel les précurseurs de PF avec DA potentiel de neurone ont d&#39;abord été générés par l&#39;activation de la sonic hedgehog et canoniques voies de signalisation Wnt au cours de la phase précoce de la différenciation, puis ces cellules PF ont été précisées pour les neurones dopaminergiques. Bien que ce protocole est plus efficace, il ya encore quelques problèmes; par exemple, l&#39;ensemble du processus de différenciation prend beaucoup de temps (au moins 35 jours) et de cellules nourricières est dépendante 15, ou EB est dépendante de l&#39;identité ou de 16 A9 14 n&#39;a pas été démontrée. Ici, sur la base de la connaissance de in vivo développement embryonnaire DA neurone et les résultats publiés d&#39;autres chercheurs de, nous avons optimisé les conditions de culture pour l&#39;effgénération isant des neurones dopaminergiques de deux CSEh et hiPSCs. Nous avons généré des cellules précurseurs première FP par l&#39;activation de la signalisation Wnt canonique avec une petite molécule et CHIR99021 signalisation Sonic Hedgehog avec de petites molécules et purmorphamine SAG. Ces cellules expriment PF FOXA2, Lmx1a, CORIN, OTX2 et nestine. Nous avons ensuite précisé ces cellules PF à neurones dopaminergiques avec des facteurs de croissance, y compris BDNF, GDNF, etc. Les neurones dopaminergiques sont générés de A9 type de cellule car ils sont positifs pour GIRK2 tout négatif pour Calbindin 17. Ce protocole est une cellule d&#39;alimentation ou EB indépendante, hautement efficace et reproductible. En utilisant ce protocole, on peut dériver neurones DA en moins de 4 semaines à partir de CSEh ou hiPSCs des personnes normales pour l&#39;étude de la transplantation de cellules, ou de hiPSCs de patients parkinsoniens pour la modélisation in vitro de PD ou de tester des agents thérapeutiques potentiels pour PD.

<table border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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.

Un aperçu du protocole de différenciation est représenté sur la Figure 1. L&#39;efficacité du protocole de différenciation présentée ici se fonde sur l&#39;état des cellules de départ. Par conséquent, il est essentiel de faire en sorte que, tout d&#39;abord une supprime toutes les colonies différenciées avant dissociation hPSCs dans des cellules individuelles de la différenciation, et d&#39;autre part, une épuise la plupart, si pas toutes, les cellules nourricières MEF par l&#39;incubati...

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.

Réalisé neurone dopaminergique différenciation de cellules pluripotentes humaines souches

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

16.4K vues.  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.

Vidéo: 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.

Dérivation efficace des ressources humaines progéniteurs neuronaux et des neurones à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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

Recherche

Neurosciences

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.

La spécification des neurones glutamatergiques télencéphaliques de cellules souches pluripotentes humaines

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.

Une méthode d&#39;extension cGMP applicable pour les agrégats de neurones humains des cellules souches et progénitrices dérivées de cellules souches pluripotentes ou fœtale tissu cérébral

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.

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

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-différenciation 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 ...

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.

Différenciation neuronale des cellules souches embryonnaires de souris 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.

Dérivation, expansion, cryopréservation et caractérisation des cellules endothéliales microvasculaires cérébrales à partir de cellules souches pluripotentes induites par l’homme

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.

Génération de neurones humains et d’oligodendrocytes à partir de cellules souches pluripotentes pour la modélisation des interactions neurone-oligodendrocytes

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.

Cellules humaines de type microglie : différenciation à partir de cellules souches pluripotentes induites et dosage in vitro de la phagocytose sur cellules vivantes à l’aide de synaptosomes humains

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.

Cultures organotypiques du cortex humain adulte comme modèle ex vivo pour la transplantation et la validation de cellules souches humaines

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