skin-punch-biopsy-explant-culture-for-derivation-primary-human

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Parkinson's Institute

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.

La diff&eacute;renciation in vitro efficace dans les types sp&eacute;cifiques de cellules est plus importante que jamais apr&egrave;s la perc&eacute;e en reprogrammation nucl&eacute;aire de cellules somatiques et son potentiel pour la mod&eacute;lisation de maladies et de d&eacute;pistage des drogues. Les facteurs cl&eacute;s de succ&egrave;s pour la diff&eacute;renciation neuronale sont le rendement de l&#39;expression du marqueur neuronal d&eacute;sir&eacute;e, la reproductibilit&eacute;, longueur et co&ucirc;t. Trois approches principales de la diff&eacute;renciation neuronale sont la diff&eacute;renciation neuronale induite par le stroma, la diff&eacute;renciation de corps embryo&iuml;des (EB) et la diff&eacute;renciation neuronale directe. Nous d&eacute;crivons ici notre protocole de neurodifferentiation &agrave; l&#39;aide de petites mol&eacute;cules qui favorisent tr&egrave;s efficacement l&#39;induction neurale dans un protocole EB 5-&eacute;tape de six lignes de (iPSC) des cellules souches pluripotentes induites de patients atteints de la maladie de Parkinson et les contr&ocirc;les. Ce protocole g&eacute;n&egrave;re des pr&eacute;curseurs neurones &agrave; l&#39;aide de Dorsomorphin et de SB431542 et de plus de maturation dans les neurones dopaminergiques en rempla&ccedil;ant sonic hedgehog avec purmorphamine ou agoniste liss&eacute;. L&#39;avantage de cette approche est que toutes les lignes sp&eacute;cifiques &agrave; un patient iPSC test&eacute;s dans cette &eacute;tude ont &eacute;t&eacute; cajol&eacute;s, syst&eacute;matiquement et avec succ&egrave;s, dans la lign&eacute;e de neurones.

Petites mol&eacute;cules am&eacute;liorent grandement la conversion des cellules souches pluripotentes induites par l&#39;homme &agrave; la lign&eacute;e neuronale.

Efficiente differenziazione in vitro in tipi cellulari specifici &egrave; pi&ugrave; importante che mai dopo la svolta in riprogrammazione nucleare di cellule somatiche e le sue potenzialit&agrave; per la modellazione di malattia e screening di stupefacenti. Fattori chiave di successo per la differenziazione neuronale sono il rendimento dell&#39;espressione desiderata marker neuronale, riproducibilit&agrave;, lunghezza e costo. Tre approcci principali differenziazione neuronale sono differenziazione neuronale indotta da stromale, differenziazione embryoid body (EB) e la differenziazione neuronale diretto. Qui, descriviamo il nostro protocollo di neurodifferentiation utilizzando piccole molecole che promuovono in maniera molto efficiente ad induzione neurale in un protocollo EB 5-stadio da sei linee di cellule staminali (iPSC) pluripotenti indotte da pazienti con malattia di Parkinson e controlli. Questo protocollo genera precursori neurali utilizzando Dorsomorphin e SB431542 e ulteriore maturazione in neuroni dopaminergici sostituendo sonic hedgehog con purmorphamine o agonista lisciato. Il vantaggio di questo approccio &egrave; che tutte le linee di iPSC paziente-specifici, testate in questo studio sono stati correttamente e costantemente blandite in lignaggio neurale.

Malini Vangipuram

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