This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006; SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry. We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work.

1. Cultura neuronale Nota: riprogrammazione dei fibroblasti in cellule staminali pluripotenti, l&#39;impegno per il lignaggio telencefalo dorsale, derivazione, amplificazione, e delle banche dei progenitori tardivi corticali (LCP) sono state descritte in Boissart et al 4. La differenziazione neuronale di cellule LCP-simile è stato effettuato anche secondo Boissart et al 4 con lievi modifiche. Sono state sviluppate altre procedure per la riprogrammazione diretta dei fibroblasti in cellule staminali pluripotenti indotte seguita dalla loro differenziazione in neuroni. Questo protocollo è stato mantenuto poiché consente la produzione selettiva di neuroni glutamatergici piramidali. <ol><li> Trattare 6 pozzetti piastre di coltura con vetrini con poli-ornitina (diluito a 1/6 in DPBS, concentrazione della 0,01%) O / N, seguita da tre lavaggi in DPBS. Quindi aggiungere laminina (concentrazione magazzino 1 mg / ml, diluito 500 volte in DPBS) per almeno 10 ore sotto cappa a flusso<html> . </li><li> Piatto e inviare NSC a bassa densità (50.000 cellule / cm 2) a 6 pozzetti piastre di coltura con vetrini in 3 ml di terreno di coltura costituito da DMEM / F12 (500 ml), 2 flaconi (5 ml) di N2 supplemento, 2 flaconi (10 ml ciascuna) di supplemento B27, 10 ml di Pen-streptomicina (Penicillina = 10.000 unità / ml e streptomicina = 10.000 unità / ml), 1 ml di 2-mercaptoetanolo (Soluzione madre: 50 mm) e laminina (1 / 500), senza fattori di crescita. PUNTO CRITICO: Eseguire questo passo con attenzione con l&#39;aggiunta di cellule con movimenti lenti rotanti al fine di ridurre il clustering delle cellule. </li><li> Rimuovere il terreno di coltura. Aggiungere mezzo N2B27 fresco contenente 2 mg / ml di soluzione laminina fresco per mantenere il neurone attaccato sui vetrini ed evitare grumi. Cambia il mezzo ogni 3 giorni. Mantenere un po &#39;del fluido rimanente (200 ml) prima di aggiungere mezzo fresco per evitare la cella da essiccazione. In alternativa, procedere rapidamente e modificare il volume totale (3 ml). </li></ol>e_title &quot;&gt; 2. lentivirali trasduzione Nota: vettori lentivirali GFP sono stati gentilmente forniti dal Dr. Uwe Maskos laboratorio presso l&#39;Istituto Pasteur (Parigi) e preparati secondo il protocollo pubblicato 5. GFP è guidata dal promotore del mouse fosfoglicerato chinasi (PGK). Per questo studio, titolo virale è stato pari a 400 ng / ml (soluzione madre in PBS 1x). <ol><li> Trasdurre umane neuroni IPSC-derivati ​​in ogni fase di maturazione da particelle virali con l&#39;aggiunta di 1 ml di soluzione madre contenente 40 ng di GFP vettori lentivirali per la cultura (ben 6 pozzetti) e incubare per 48 ore in terreno di coltura fresco. Nota: per questo protocollo, la durata dell&#39;incubazione permette una buona etichettatura delle strutture della colonna vertebrale. </li></ol> 3. immunofluorescenza Nota: Al fine di migliorare l&#39;etichettatura di tutta la morfologia della colonna vertebrale, etichettatura immunofluorescenza è stata effettuata utilizzando un anticorpo anti-GFP in condizioni permeabilizzate. </p&gt; <ol><li> Togliere terreno di coltura e riparare cellule trasdotte su vetrini in paraformaldeide al 4% per 10 min a temperatura ambiente, poi lavare 3 volte in 1x PBS (10 minuti ciascuno). </li><li> Coprioggetto immergere in PBS integrato con 0,05% Triton (100x) e il 10% siero di cavallo per 1 ora a temperatura ambiente, poi lavare 3 volte in 1x PBS. </li><li> Aggiungere 100 pl di PBS 1x integrato con siero di cavallo 4% e anticorpo primario (1 / 1.000) sollevata contro GFP e diluito di un fattore di 1,000 su ciascun vetrino. Incubare in una scatola buia O / N a 4 ° C, quindi lavare 3 volte con PBS 1x. </li><li> Diluire Alexa Fluor 488 coniugato anticorpo (1/200) in PBS integrato con 0,5% di Tween 20 e incubare per 1 ora a RT. Quindi lavare 3 volte in PBS 1x e montare coprioggetto su vetrini con mezzo di montaggio per la microscopia a fluorescenza. </li></ol> 4. spine dendritiche Imaging <ol><li> Eseguire confocale Imaging attraverso un microscopio confocale a scansione laser. </li><li> Selezionare neuroni sani con una morfologia piramidaletrovare una dendritica pieno, e quantificare almeno 10 neuroni per condizione da esperimenti separati. Quantificare 60-100 micron per dendriti. </li><li> Acquisire le immagini utilizzando un olio 40X NA = 1.3 obiettivo e una linea laser 488 nm per GFP di eccitazione, con una potenza di picco tipica a livello di campione di circa 20? W. Imposta la dimensione dei pixel circa 80 nm per assaggiare correttamente spine dendritiche. Nota: La successiva chiamata analisi per un&#39;immagine in cui il rumore non compromette la corretta segmentazione dei dendriti e spine. Con le impostazioni di campionamento e di potenza spaziale di cui sopra, abbiamo osservato che un pixel di sosta a tempo di 3.15 ms è sufficiente per raccogliere abbastanza fotoni per costruire una tale immagine. Per campioni dim, la qualità dell&#39;immagine può essere migliorata facendo la media da 2 a 4 scansioni. L&#39;acquisizione di diverse piastrelle XY può essere necessario per coprire la regione di interesse, che poi deve essere cucita insieme prima della trasformazione. </li><li> Per campionare l&#39;intero volume neurone, acquisire un Z-stack, conZ spaziatura da 150 nm a 300 nm, producendo 20 a 30 fette Z. Nota: La risoluzione spaziale laterale realizzato con queste impostazioni è 234 nm e la risoluzione assiale è 591 nm. Il campionamento prescelto qui è adeguata, ma la risoluzione assiale è maggiore della dimensione minima delle spine da acquisire, l&#39;analisi favorisce le spine che si estendono lateralmente da dendriti. </li></ol> 5. 3D Quantificazione di spine dendritiche Nota: Le seguenti sezioni specificamente descrivono l&#39;uso del software Imaris per l&#39;analisi. Esistono implementazioni alternative, tra cui NeuronStudio 15 o Metamorph 8, che può fornire risultati simili. Utilizzare le seguenti impostazioni chiave: <ol><li> Come una fase di pre-processing, utilizzare il filtraggio gaussiana attraverso i servizi di elaborazione delle immagini offerte dal software. Eseguire il filtraggio gaussiano via Elaborazione immagini&gt; Smoocosa&gt; Filtro gaussiana. Impostare la larghezza del filtro di essere uguale alla dimensione dei pixel in XY. </li><li> Eseguire un tracciato semi-automatica dei dendriti, utilizzando il modulo Filament Tracer del software. <ol><li> Innanzitutto, stimare il diametro dendrite, utilizzando lo strumento distanza nella scheda Fetta del software. </li><li> Nella scheda Surpass, fare clic sullo strumento filamenti. Per una migliore robustezza, questo protocollo si basa su di tracking semi-automatico; clicca su Skip creazione automatica. L&#39;interfaccia del modulo ora sta mostrando la scheda Draw. Qui, seleziona AutoPath come metodo, Dendrite come tipo, e il diametro di ingresso dendrite stimato. </li><li> Utilizzare la Selezionare la modalità del puntatore, trasformando il cursore in una scatola. Shift-pulsante destro del mouse sul punto di partenza dendriti. Nota: il software esegue calcoli iniziali. </li><li> Spostare il puntatore lungo dendrite. Dal punto di partenza (rappresentareDE come una sfera blu), è mostrata una linea gialla che rappresenta il percorso dendrite più probabile. Shift-sinistra del mouse sul punto finale dendriti. </li></ol></li><li> Eseguire la spine di segmentazione automatica. Nota: Le spine sul dendrite tracciato si trovano automaticamente l&#39;interfaccia del modulo. <ol><li> Nell&#39;interfaccia del modulo, fare clic sulla scheda Creazione. Nel Ricostruisci casella di riepilogo, scegliere Rebuild Dendrite Diametro e selezionare la casella di dati Mastio. Quindi fare clic su Ricostruisci. </li><li> Impostare la soglia in modo che il volume segmentato corrispondente al volume effettivo dendrite. Come un algoritmo, selezionare più breve distanza dal Mappa distanza. Fare clic sul pulsante Avanti. </li><li> Determinare il più piccolo diametro della testa della colonna vertebrale e la lunghezza massima, sempre utilizzando lo strumento distanza nella scheda Fetta di software, per poi tornare alla scheda Surpass e immettere i parametri. Fo questo protocollo, i valori intorno a 200-300 nm per il diametro minimo e 4 micron per la lunghezza massima sono buoni punti di partenza. Non selezionare la casella Consenti Branch Spine. Fare clic sul pulsante Avanti. </li><li> Regolare la soglia Punti di semi in modo che i punti blu rappresentano spine localizzano alle teste della colonna vertebrale reali. Fare clic sul pulsante Avanti. Nota: Il calcolo nucleo è fatto ora e può essere lungo. </li></ol></li><li> Classificare spine andando alla scheda Strumenti dell&#39;interfaccia modulo. Clicca su Classifica Spine. Nell&#39;interfaccia utente viene richiesto, assicurarsi che non vi sono quattro classi, definite dalla loro morfologia come segue: tozza: lunghezza &lt;1 micron; Funghi: Lunghezza (colonna vertebrale)&gt; 3 e la larghezza massima (testa)&gt; significano larghezza (collo) x 2; Lungo e sottile: Media larghezza (testa) ≥ media larghezza (collo); Filopodia simile: Lunghezza ≤ 4 micron (senza testa). Nota: il moInterfaccia Dule genera quattro nuovi oggetti ad incandescenza contenenti i risultati della classificazione. </li><li> Esportazione Dati statistici: su uno di questi quattro interfacce degli oggetti, vai alla scheda Statistiche. Clicca sui Esporta tutte le statistiche su File. Nota: Altri valori statistici possono essere esportati per dendriti (es., Lunghezza, area, diametro, profondità ramo, diramazione angolo, volume, ecc media.), E per spine (per esempio, rettilineità, zona di attacco, la lunghezza e il volume di distinta parti della colonna vertebrale, del diametro della colonna vertebrale, densità, ecc.) </li></ol>

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The authors have nothing to disclose.

La quantificazione delle caratteristiche morfologiche di neuroni piramidali invocato il software. L&#39;interfaccia Filament Tracer è stato utilizzato per la segmentazione dei neuroni e spine, e il modulo XT è stato utilizzato per l&#39;analisi. Per analizzare la precisione della nostra tecnica, in primo luogo abbiamo confrontato i parametri morfologici misurati (lunghezza, area e volume totale della colonna vertebrale se applicabile), con quelli pubblicati utilizzando ratto neuroni pirami...

Spine dendritiche di neuroni piramidali corticali sono piccoli e sottili protuberanze che sono distribuiti lungo i dendriti basali e apicali di questi sottotipi neuronali in roditori, primati, e il cervello umano. Sono i luoghi di maggior sinapsi eccitatorie e visualizzare funzioni chiave nell&#39;apprendimento e processi cognitivi. Le strutture dettagliate delle spine dendritiche umani sono stati tecnicamente studiati da microscopia elettronica 2. Tuttavia, tale approccio è che richiede tempo e rappresenta pesante carico di lavoro. Più recentemente, un tridimensionale (3D) ricostruzione della morfologia delle spine dendritiche è stata riportata nella corteccia cerebrale umana usando software specifico combinato a grandi analisi colonna 3 manuale. Tecnologia verde proteina fluorescente (GFP) accoppiato ad immunofluorescenza rappresenta uno strumento preciso per l&#39;identificazione della colonna vertebrale e la misurazione forma al microscopio a fluorescenza. Questo approccio può essere facilmente applicato a colture neuronali. However, i dati non sono stati riportati sull&#39;analisi di maturazione della colonna vertebrale e della morfologia dei neuroni umani derivate da cellule staminali pluripotenti indotte (IPSC). L&#39;obiettivo di questo studio è stato quello di descrivere un protocollo, che permette di spine dendritiche di imaging da colture di neuroni umani in vitro. Etichettatura GFP, microscopia confocale e analisi 3D con il modulo Filament Tracer di software Imaris sono stati utilizzati nel presente protocollo. Cultura passi che sono necessari per ottenere i neuroni glutamatergici corticali degli strati II-IV da cellule staminali neurali (NSC) sono anche brevemente descritto qui. L&#39;intero protocollo per la produzione NSC umano è già stato pubblicato altrove 4.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PD-PBS (1X), sans Calcium, Magnesium et Phenol Red</td>
			<td>Gibco/ Life Technologies</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine Solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin</td>
			<td>Dutscher Dominique</td>
			<td>354232</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco/ Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement w/o vit A (50X)</td>
			<td>Gibco/ Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/NUT.MIX F-12 W/GLUT-I</td>
			<td>Gibco/ Life Technologies</td>
			<td>31331028</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Med SFM</td>
			<td>Gibco/ Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco/ Life Technologies</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Steptomycin</td>
			<td>Gibco/ Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Rabbit Serum Polyclonal Antibody</td>
			<td>Gibco/ Life Technologies</td>
			<td>A-6455</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco/ Life Technologies</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Rabbit&#160;</td>
			<td>Gibco/ Life Technologies</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Anti-betaIII tubulin antibody</td>
			<td>Millipore</td>
			<td>AB9354</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass 13 mm</td>
			<td>VWR</td>
			<td>631-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent avec DAPI</td>
			<td>Gibco/ Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween(R) 20 Bioextra, Viscous Liquid</td>
			<td>Sigma Aldrich Chimie</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich Chimie</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Fibroblasts</td>
			<td>Coriell Cell Line Biorepository</td>
			<td>GM 4603 and GM 1869</td>
			<td>Coriell Institute for Medical Research, Camden, NJ, USA</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss (Germany)</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris Software</td>
			<td>Bitplane AG, Zurich</td>
			<td>6.4.0 version</td>
			<td>Filament Tracer and Imaris XT modules are necessary</td>
		</tr>
		<tr>
			<td>Huygens Software</td>
			<td>Huygens software, SVI, Netherlands</td>
			<td>Pro version</td>
			<td>Optional (for deconvolution testing)</td>
		</tr>
	</tbody>
</table>

three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

Spine dendritiche sono piccole sporgenze che corrispondono ai compartimenti post-sinaptici di sinapsi eccitatorie nel sistema nervoso centrale. Essi sono distribuiti lungo i dendriti. La loro morfologia dipende in gran parte l&#39;attività neuronale, e sono dinamici. Spine dendritiche esprimono i recettori glutamatergici (recettori AMPA e NMDA) sulla loro superficie e ai livelli di densità postsinaptiche. Ogni colonna vertebrale permette al neurone di controllare la propria attività statali e locali in modo indipendente. Morfologie della colonna vertebrale sono stati ampiamente studiati in cellule piramidali glutammatergici della corteccia cerebrale, utilizzando entrambi gli approcci in vivo e colture neuronali ottenute da tessuti di roditori. Condizioni neuropatologiche possono essere associate all&#39;induzione colonna vertebrale alterata e maturazione, come mostrato in roditori neuroni in coltura e analisi quantitativa unidimensionale 1. Il presente studio descrive un protocollo per l&#39;analisi quantitativa 3D di morfologie della colonna vertebrale utilizzando cortic umanoAl neuroni derivati ​​da cellule staminali neurali (progenitrici fine corticali). Queste cellule sono state inizialmente ottenute da cellule staminali pluripotenti indotte. Questo protocollo permette l&#39;analisi di morfologie della colonna vertebrale in periodi diversi della cultura, e con possibilità di confronto tra le cellule staminali pluripotenti indotte ottenuti da individui di controllo con quelli ottenuti da pazienti con malattie psichiatriche.

<html> Il presente studio descrive un protocollo standardizzato per la quantificazione della colonna vertebrale dei dendriti coltura di neuroni piramidali derivati ​​da IPSC. Questo protocollo permette l&#39;analisi della colonna maturazione sui neuroni umani e la sua possibile confronto con la maturazione delle spine in colture neuronali roditore standard così come in vivo in modelli animali. Figura 1A rappresenta uno schema delle varie fasi di coltura che perm...

Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Spine dendritiche di neuroni piramidali sono i siti di maggior sinapsi eccitatorie in mammiferi corteccia cerebrale. Il metodo descrive un&#39;analisi quantitativa 3D di morfologie della colonna vertebrale nei neuroni piramidali glutamatergici corticali umane derivate da cellule staminali pluripotenti indotte.

Quantificazione tridimensionale di spine dendritiche da piramidale neuroni derivate da cellule staminali umane pluripotenti indotte

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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Research

JoVE Journal

Developmental Biology

11.3K Views.  Institut Pasteur.  Spine dendritiche di neuroni piramidali sono i siti di maggior sinapsi eccitatorie in mammiferi corteccia cerebrale. Il metodo descrive un'analisi quantitativa 3D di morfologie della colonna vertebrale nei neuroni piramidali glutamatergici corticali umane derivate da cellule staminali pluripotenti indotte. 

Video: Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Ricerca

Biologia dello sviluppo

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Derivazione senza alimentatore di melanociti da cellule staminali umane pluripotenti

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Generazione di cellule staminali pluripotenti indotte da melanoma umano Linfociti infiltranti il ​​tumore

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

Differenziare i condrociti da cellule staminali pluripotenti indotte dall&#39;uomo derivato dal sangue

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Formazione di pellet condensato da cellule staminali pluripotenti indotte dal sangue

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Microcircuito sinaptica modellazione con 3D Cocultures di astrociti e neuroni da cellule staminali pluripotenti umane

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...