The authors have no conflicts of interest to disclose.

1. Arricchimento di emopoietiche progenitrici cellule da sangue intero per adulti NOTA: le cellule progenitrici ematopoietiche o cellule CD34 + possono essere purificati da cellule mononucleate del sangue periferico (PBMC) derivate da una varietà di fonti, tra cui il sangue del cordone, materiale leucaferesi e sangue intero con metodi basati gradiente di densità di centrifugazione. Il metodo qui elencati utilizza sangue intero come esempio. <ol><li> Isolamento di PBMC da sangue intero: <ol><li> Aggiungere 4,5 ml di mezzo di separazione dei linfociti al tubo SepMate pipettando attraverso il foro centrale dell&#39;inserto. </li><li> Diluire il campione di sangue con un uguale volume di DPBS sterili contenenti siero umano 2% (v / v). Ad esempio, diluire 5 ml di campione con 5 ml di siero umano DPBS + 2%. </li><li> Aggiungere il campione diluito pipettando giù il lato del tubo. Fare attenzione a non versare il campione direttamente attraverso il foro centrale. </li><li> Centrifugare a 1200 xg per 10 minuti a RT. </li><li> Rimuovere con attenzione il surnatante sopra il livello PMBC. </li><li> Raccogliere lo strato PBMC (nuvoloso) in un nuovo tubo. </li><li> Aggiungere 15 ml di fosfato di Dulbecco tamponata Saline (DPBS) contenente siero umano 2% nel tubo e centrifugare a 150 xg per 10 min a RT con il freno off. </li><li> Eliminare il supernatante e risospendere il pellet in 15 ml di DPBS contenente siero umano 2%. Contare il numero di cellule. </li><li> Centrifugare a 690 xg per 10 min a RT. Rimuovere tutto il surnatante dal tubo. </li><li> Risospendere le cellule a 1 x 10 7 cellule per 15 ml di medium (IMDM) Dulbecco modificato di Iscove contenente 1% di antibiotici (v / v) e siero umano al 10% (v / v). </li><li> Incubare le cellule O / N a 37 ° C in 5% CO 2 incubatore prima isolare cellule CD34 +. </li></ol></li><li> Selezione positiva di cellule CD34 + da PBMC utilizzando CD34 Multisort Kit: NOTA: lavoro veloce e utilizzare soluzioni pre-raffreddato a rendere le cellule freddo e prevenire tappaturadi anticorpi sulla superficie cellulare e l&#39;etichettatura di cellule non-specifico per l&#39;istruzione kit. <ol><li> Raccogliere tutti PMBCs in un tubo da 50 ml e contare il numero di cellule totali nel mezzo. </li><li> Centrifugare le cellule a 300 xg per 10 min. Rimuovere completamente tutto il surnatante. </li><li> Risospendere 8 fino a 10 300 cellule per ml in tampone tallone (DPBS contenente 0,5% di siero umano (v / v) e 2 mM EDTA). </li><li> Aggiungere 100 ml di FcR reagente di bloccaggio e aggiungere 100 ml di MultiSortMicroBeads CD34. </li><li> Mescolare bene e incubare per 30 minuti in frigorifero (2 - 8 ° C). </li><li> Lavare le cellule aggiungendo 2 ml di tampone per tallone 10 8 cellule e centrifugare a 300 xg per 10 min a RT. Rimuovere completamente il surnatante. </li><li> Risospendere fino a 10 8 cellule in 500 microlitri di tampone. </li><li> Inserire colonna MACS LS nel campo magnetico di un separatore MACS e preparare colonna risciacquando con 3 ml di tampone tallone. </li><li> Applicare sospensione cellulare sul center della colonna. Cellule senza etichetta fluiranno attraverso cui possono essere raccolti, se si preferisce. </li><li> Lavare colonna tre volte con 3 ml di tampone tallone. </li><li> Rimuovere la colonna dal separatore e posizionarlo su un tubo 15 ml. </li><li> Aggiungere 5 ml di tampone tallone nella colonna e lavare immediatamente le cellule marcate magneticamente spingendo lo stantuffo nella colonna. </li><li> Aggiungere 20 ml di MULTISORT Reagente di uscita per 1 ml di sospensione cellulare. Mescolare bene e incubare per 10 minuti in frigorifero (2 - 8 ° C). </li><li> Aggiungere 1 - 2 ml di tampone tallone per 10 7 cellule e centrifugare le cellule a 300 xg per 10 min a RT. Rimuovere completamente il surnatante. </li><li> Risospendere le cellule in 50 ml di tampone tallone per 10 7 cellule. </li><li> Aggiungere 30 ml di MULTISORT arresto reagente per 10 7 cellule e incubare per 15 minuti in frigorifero. </li><li> Aggiungere 5 ml di tampone tallone e centrifugare le cellule a 300 xg per 10 min a RT. Scartare il surnatante. </li></ol></li></ol> 2. Derivazione di cellule staminali neurali indotte da cellule CD34 + <ol><li> Cellule CD34 + cultura: NOTA: media appena preparato deve essere utilizzato entro 7 giorni se conservata in frigorifero. Significativa perdita di cellule CD34 può essere osservata dopo 24 ore, ma la significativa proliferazione delle cellule dovrebbe essere evidente, soprattutto dopo giorno 5. Se il numero di cellule diminuisce continuamente o non c&#39;è proliferazione, esso implica o cattiva qualità delle cellule medie o CD34 e cellule non deve essere utilizzato per il passo successivo. <ol><li> Risospendere e cultura CD34 + cellule CD34 mantenendo medio (StemSpan SFEM terreno contenente trombopoietina umana (TPO, 100 ng / ml), FMS-come tirosin chinasi 3 (Flt-3) ligando (100 ng / ml) e fattore delle cellule staminali (SCF, 100 ng / ml), interleuchina-6 (IL-6, 20 ng / ml) e interleuchina-7 (IL-7, 20 ng / ml)) in una piastra a 24 pozzetti (fino a 3 x 10 5 / pozzetto in 1 ml di terreno per pozzetto) a 37 ° C in 5% CO 2 incubatore. </li><li> After 24 ore, raccogliere le cellule galleggianti e centrifugare le cellule a 110 xg a temperatura ambiente per eliminare eventuali cellule collegate e detriti cellulari. Eliminare il supernatante e risospendere le cellule in 1 ml di CD34 mantenere fresco medio e seme in una nuova piastra 24 pozzetti (fino a 1 x 10 5 / pozzetto) a 37 ° C in 5% CO 2 incubatore per 5 giorni aggiuntivi. </li><li> Sostituire la metà del mezzo ogni l&#39;altro giorno aspirando accuratamente la metà superiore surnatante mentre le cellule CD34 + sono galleggianti sul fondo dei pozzetti (Figura 1A). </li></ol></li><li> Sendai trasfezione virus: <ol><li> Il giorno 5, raccogliere le cellule CD34 e centrifugare le cellule a 170 xg per 10 min a RT. Scartare il surnatante. </li><li> Risospendere le cellule in terreno fresco a 1 x 10 5 / pozzetto in una piastra da 24 pozzetti in 1 ml di terreno. </li><li> Scongelare un kit riprogrammazione cytotune-iPS Sendai dapprima immergendo il fondo dei tubi in un bagno di 37 ° C l&#39;acqua per 10 secondi e poi completamente scongelare unt RT. Brevemente centrifugare le provette e metterli su ghiaccio. Preparare l&#39;impasto Sendai miscelando il volume consigliato di ogni virus elencati sul certificato di analisi (COA) per il lotto corrispondente. MOI 15 è raccomandato. </li><li> Aggiungere mix Sendai a ciascun pozzetto di cellule CD34. Mescolare i pozzetti scuotendo delicatamente. Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Osservare l&#39;efficienza di trasfezione dopo 24 ore. Si noti che aggregati di cellule e la formazione sfera sono indicatori per trasfezione di successo (Figura 1B). </li><li> Cambiare la metà del mezzo trasferendo accuratamente la metà superiore del mezzo ad un altro bene. Se ci sono sfere cellule nel nuovo pozzo, aggiungere la stessa quantità di mezzo fresco. </li><li> Modificare media ogni l&#39;altro giorno ripetendo il passaggio 2.2.6. </li></ol></li><li> Induzione di cellule staminali neurali: NOTA: A seconda delle condizioni delle cellule, circa 5 o 7 giorni dopo la trasfezione, le cellule aderenti monostrato raggiungeranno 30 -50% di confluenza (Figura 1C). <ol><li> Rimuovere il surnatante contenenti celle galleggianti e sfere e quindi aggiungere 1 ml di terreno progenitrici neurali (Dulbecco Modified Eagle Medium: miscela di nutrienti F-12 (DMEM / F12), contenente 1x N2 supplemento, 0,1% (w / v) di albumina sierica bovina ( BSA), 1% (v / v) antibiotici, 20 ng / ml di fattore di crescita basico dei fibroblasti (bFGF) e 20 ng / ml di fattore di crescita epidermico (EGF)) nelle cellule aderenti. </li><li> Facoltativamente, dissociare le sfere cellule pipettando delicatamente e centrifugare le cellule a 170 xg per 10 min. Rimuovere il surnatante e risospendere le cellule in 1 ml di terreno progenitrici neurali in una nuova piastra 24 pozzetti rivestite con matrigel per istruzione come backup. </li><li> Modificare il supporto a giorni fino alle cellule raggiungono 60 - 80% confluenti, di solito dopo 1 settimana. </li><li> Eliminare il surnatante e aggiungere 1 ml di terreno di cellule staminali neurali (media siero libero, NSC SFM). Dissociare il celle utilizzando un raschietto cellulare seguitada molto delicato pipettaggio. </li><li> Rimuovere le cellule e sementi tutti in un pozzetto di una piastra da 6 pozzetti. Aggiungi un altro 1 ml di mezzo di cellule staminali neurali per fare 2 ml supporto per ciascun bene. Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Modificare media ogni l&#39;altro giorno. </li><li> Quando le cellule hanno raggiunto il 60% di confluenza, dissociarsi e replate le cellule a rapporto 1: 3 seguendo la procedura descritta in 2.3.4 e 2.3.6. </li></ol></li><li> Congelamento delle cellule staminali neurali: <ol><li> Preparare cellule staminali neurali congelamento mezzo aggiungendo 20% dimetilsolfossido (DMSO) nel terreno di cellule staminali neurali. Mantenere il mezzo congelamento su ghiaccio. </li><li> Dissociano le cellule in un 6-pozzetti, utilizzando raschietto cella seguita pipettando delicatamente. Contare le cellule. Centrifugare le cellule a 170 xg per 10 min a RT. Scartare il surnatante. </li><li> Risospendere le cellule in terreno cellule staminali neurali a 1 x 10 6 / ml. Aggiungere lo stesso volume di congelamento mezzo alle cellule. </li><li> Aggiungere 1 ml (5 x 10 5 cellule totali) di cellule in un esageratamente. Congelare le cellule utilizzando un congelamento contenitore Mr. Froster seguendo le istruzioni. </li></ol></li></ol> 3. Neural differenziazione cellulare <ol><li> Cellule neuronali differenziazione: <ol><li> Quando le cellule staminali neurali raggiungono l&#39;80% di confluenza, staccare le cellule staminali neurali con un poliziotto di gomma e poi dissociarsi pipettando delicatamente. </li><li> Contare le cellule e regolare la concentrazione di 1 x 10 5 / ml in mezzo di cellule staminali neurali. </li><li> Per la differenziazione neuronale, piastra le cellule sulla copertura poli-D-lisina / laminina rivestito scivola in piastre da 24 pozzetti a 1 x 10 5 / bene. Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Sostituire il supporto con il mezzo differenziamento neurale (DMEM / F12 contenenti 1x N2 supplemento, 1x B27 supplemento, 300 ng / ml cAMP, 0,2 mm di vitamina C, di 10 ng cervello ml / derived neurotrophic factor (BDNF) e 10 ng / ml cellule gliali derivati fattore neurotrofico (GDNF)) after 24 ore. Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Cambiare terreno di differenziamento neurale due volte alla settimana per 2 settimane. </li></ol></li><li> Differenziazione astrogliali: <ol><li> Dissociare le cellule staminali neurali pipettando. </li><li> Contare le cellule e regolare la concentrazione cellulare di 5 x 10 4 cellule / ml. Seme le cellule su piastre da 24 pozzetti. Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Sostituire il mezzo con DMEM / F12 con siero fetale bovino 10% (FBS) e incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Cambiare media due volte alla settimana per 2 settimane. Se le cellule raggiungono confluenza prima di 2 settimane replate le cellule seguenti operazioni 3.2.1 e 3.2.2. </li></ol></li><li> Oligodendrocyte differenziazione: <ol><li> Dissociare le cellule staminali neurali pipettando. </li><li> Contare le cellule e di semi 1 x 10 5 cellule in piastre da 24 pozzetti rivestiti di poli-L-Ornitina in 1 ml di oligodendrociti Differentimedia zione costituito da DMEM / F12 contenenti supplemento N2, 10 ng / ml di piastrine derivato fattore di crescita-AA (PDGF-AA), 2 ng / ml di neurotrofina-3 (NT-3), 2 ng / ml di sonic hedgehog ( Shh), e 3 Nm di triiodotironina (T3). Incubare le cellule a 37 ° C in 5% CO 2 incubatore. </li><li> Cambiare media due volte alla settimana per 2 settimane. </li><li> Sostituire il mezzo con DMEM / F12 con 1xN2 supplemento e 3 Nm di T3 e di cultura le cellule per una settimana supplementare per proteina basica della mielina (MBP) produzione. </li></ol></li></ol> 4. Immunofluorescenza <ol><li> Sostituire media con 4% paraformaldeide (PFA). Incubare per 10 minuti a temperatura ambiente. </li><li> Scartare PFA e lavare con PBS contenente 0,1% Triton X-100 (PBS-T) per 3 volte, 5 min ciascuna volta. </li><li> Permeabilize cellule con PBS contenente 0,5% Triton X-100 per 10 minuti a RT. </li><li> Bloccare le cellule con PBS-T contenente siero di capra 4% per 20 minuti a RT. </li><li> Incubare le cellule con corrispondenti Antibo primariostampi diluito in PBS-T contenente 0,1% BSA per 2 ore a temperatura ambiente o O / N in cella. </li><li> Lavare le cellule per 3 volte con PBS-T, 5 min ciascuna volta. Incubare le cellule con corrispondenti anticorpi secondari accoppiati con un fluorocromo utilizzando 1: 400 di diluizione in PBS-T contenente 0,1% BSA per 1 ora su un agitatore al buio. </li><li> Lavare le cellule con PBS-T contenente 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) per 10 minuti a RT. </li><li> Lavare le cellule due ulteriori volte con PBS-T per 5 minuti ciascuno a RT. </li><li> Osservare le cellule marcate con un microscopio e scattare foto (figura 3). </li></ol>

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The study is funded by NIH intramural funds.

Viene fornito un protocollo dettagliato per generare direttamente le cellule staminali neurali da cellule progenitrici ematopoietiche periferiche. Rispetto alle cellule di fibroblasti, sangue periferico umano è più accessibile. Utilizzando il protocollo presentato, più di 1 x 10 5 cellule progenitrici ematopoietiche o cellule CD34 positive, può essere arricchito da 10 ml di sangue intero. Sebbene contaminazione con piastrine di solito non è evitabile, può essere facilmente r...

In vitro colture neuronali sono state utilizzate come strumento fondamentale per lo studio di malattie neurologiche. Animale primario (principalmente roditori) colture neuronali 1, 2 e linee cellulari neuronali umane derivate da gliomi o altri tumori sono i più comunemente utilizzati in tali studi. Tuttavia, è stato riconosciuto che non vi sono differenze significative tra roditori e cellule umane. Molte scoperte basate su roditori non possono essere tradotti per gli esseri umani. Inoltre, con i rapidi sviluppi l&#39;analisi delle informazioni di massa genomica e la relativamente facile editing gene e sequenziamento dell&#39;intero genoma, la tendenza è sempre più orientata alla scoperta di malattia geni inclini e delineare le loro funzioni e ruoli a malattie specifiche, il che rende i pochi neuronale umana linee cellulari solo hanno limitato l&#39;utilizzo. Teoricamente, colture neuronali primarie umane derivate da campioni di sistema nervoso paziente sono la scelta migliore ma sono impossibili da ottenere; meth quindi alternativaODS sono necessari. Negli ultimi anni, alcuni approcci sono stati perseguiti, con due è il più distinguibili. Dopo lo sviluppo della tecnica di generare cellule staminali pluripotenti indotte (IPSC) utilizzando il mouse e cellule somatiche umane 3, 4, cellule neurali potrebbe essere ulteriormente differenziate da loro 5-7. Tuttavia, la generazione e la caratterizzazione iPSC richiede intensità di lavoro, le tecniche, e l&#39;ingresso di tempo, a volte anche eccessivamente. Poco dopo, un altro approccio è stato sviluppato per trasformare direttamente le cellule neuronali dalle cellule somatiche 8, 9. Poiché i neuroni risultanti sono non-proliferativa, limita la sua applicazione in studi intensivi e screening di farmaci, che richiede una grande quantità di cellule. Per prendere i vantaggi di entrambe le tecniche, diretta derivazione di cellule staminali neurali / cellule progenitrici a partire da cellule somatiche è stata esplorata da diversi gruppi di 10-12, che bypassa il tedioso processo di generazione iPSC e caratterizzazione, ma comunque provides un numero decente di cellule staminali neurali per dopo differenziamento neurale. Abbiamo precedentemente dimostrato che in seguito all&#39;introduzione dei fattori di trascrizione Yamanaka in cellule progenitrici ematopoietiche, cellule staminali neurali possono essere generati direttamente utilizzando una cellula progenitrice neurale selezionando media 13. Qui riportiamo il metodo in dettaglio.

<table border="0" cellpadding="0" cellspacing="0" width="584">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Material List</td>
			<td style="width:171px;"></td>
			<td style="width:135px;"></td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:72px;">Comments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Lymphocyte Separation Medium</td>
			<td style="width:171px;">Lonza</td>
			<td style="width:135px;">17-829E</td>
			<td style="width:72px;">1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SepMate</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">15415</td>
			<td style="width:72px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Human Serum</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:135px;">34005-100</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Antibiotics</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:135px;">15240-062</td>
			<td align="right" style="width:72px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CD34 MultiSort Kit</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-056-701</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EDTA</td>
			<td style="width:171px;">Cellgro</td>
			<td style="width:135px;">46-034-Cl&#160;</td>
			<td style="width:72px;">2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">MACS LS Column</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-042-401</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">StemSpan SFEM Medium</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">9650</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IMDM</td>
			<td style="width:171px;">Quality Biologicals</td>
			<td style="width:135px;">112-035-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TPO</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-18</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Flt-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-19</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SCF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-07</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-6</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-06</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-7&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-07</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IPS Sendai Reprogramming Kit</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">A1378001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Cell scraper</td>
			<td style="width:171px;">Sarstedt</td>
			<td align="right" style="width:135px;">83.183</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMSO</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D2650</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CryoTube vials</td>
			<td style="width:171px;">Thermo</td>
			<td align="right" style="width:135px;">368632</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Mr. Frosty container</td>
			<td style="width:171px;">Thermo</td>
			<td style="width:135px;">5100-0001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMEM/F12</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">12400-024</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">N2 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A2934</td>
			<td style="width:72px;">0.1% (w/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">bFGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-18B</td>
			<td style="width:72px;">20 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">AF-100-15</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">B27 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17504-044</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NSC serum free medium</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:135px;">A1050901</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="40">
			<td height="43" rowspan="2" style="height:43px;width:208px;">Poly-D-lysine/laminin coated cover slips</td>
			<td rowspan="2" style="width:171px;">BD Bioscences</td>
			<td align="right" rowspan="2" style="width:135px;">354087</td>
			<td rowspan="2" style="width:72px;"></td>
		</tr>
		<tr height="3">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">cAMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A9501</td>
			<td style="width:72px;">300 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Vitamin C</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A0278</td>
			<td style="width:72px;">0.2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-02</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">GDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-10</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P4957</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PDGF-AA</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-13A</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NT-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-03</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Shh</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">1314-SH/CF</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">T3</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T6397</td>
			<td style="width:72px;">3 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PFA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:208px;">PBS</td>
			<td style="width:171px;">Quality Biological</td>
			<td style="width:135px;">119-069-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Goat serum</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G9023</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TritonX-100</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T9284</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse monoclonal anti-Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:135px;">AB5922</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-SOX2&#160; antibody</td>
			<td style="width:171px;">Applied Stemcell</td>
			<td style="width:135px;">ASA0120</td>
			<td style="width:72px;">Ready to use</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse anti-&#946;III-tubulin antibody</td>
			<td style="width:171px;">Promega</td>
			<td style="width:135px;">G712A</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:208px;">Rabbit anti-GFAP antibody</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G4546</td>
			<td style="width:72px;">1:100 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-O4 antibody</td>
			<td style="width:171px;">R&#38;D Systems</td>
			<td style="width:135px;">MAB1326</td>
			<td style="width:72px;">IgM; 1 ng/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 594 goat anti-rabbit antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11012</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 488 goat anti-mouse antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11001</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Alexa Fluor 488 goat anti-mouse IgM antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A21042</td>
			<td style="width:72px;">1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DAPI</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D9542</td>
			<td style="width:72px;">1 ug/ml</td>
		</tr>
	</tbody>
</table>

direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells

Malattie umane specifiche colture neuronali sono essenziali per la generazione di modelli in vitro per le malattie neurologiche umane. Tuttavia, la mancanza di accesso alle adulti umano colture neuronali primarie pone sfide uniche. I recenti sviluppi in cellule staminali pluripotenti indotte (IPSC) fornisce un approccio alternativo per derivare culture neuronali di fibroblasti cutanei attraverso specifico paziente iPSC, ma questo processo è laborioso, richiede competenze specifiche e una grande quantità di risorse, e può richiedere diversi mesi. Ciò impedisce l&#39;ampia applicazione di questa tecnologia per lo studio delle malattie neurologiche. Per superare alcuni di questi problemi, abbiamo sviluppato un metodo per ottenere cellule staminali neurali direttamente dal sangue periferico umano adulto, bypassando il processo di derivazione IPSC. Cellule progenitrici ematopoietiche arricchite da sangue periferico umano adulto sono state coltivate in vitro e trasfettate con vettori Sendai contenenti fattori di trascrizione Sox2, ottobre3/4, Klf4, e c-Myc. I risultati di trasfezione di cambiamenti morfologici nelle cellule che vengono ulteriormente selezionati utilizzando media progenitrici neurali umane contenenti base del fattore di crescita dei fibroblasti (bFGF) e fattore di crescita vascolare endoteliale (VEGF). Le cellule risultanti sono caratterizzate dall&#39;espressione di marcatori di cellule staminali neuronali, come nestin e SOX2. Queste cellule staminali neurali potrebbero essere ulteriormente differenziati ai neuroni, astroglia e oligodendrociti in mezzi differenziazione specificato. Usando campioni di sangue periferico umano facilmente accessibili, questo metodo potrebbe essere utilizzato per ottenere cellule staminali neurali per un&#39;ulteriore differenziazione di cellule neurali per la modellazione in vitro di disturbi neurologici e può avanzare studi relativi alla patogenesi e il trattamento di queste malattie.

<html> La qualità delle cellule CD34 è critico per il successo di trasduzione di cellule staminali neurali. Cellule CD34 alta qualità proliferano durante il primo paio di giorni di coltura e appaiono come non aderenti, cellule omogeneamente rotonde, galleggiante appena sopra il fondo di recipienti di coltura (Figura 1A). I risultati infettati in aggregati di cellule, che si espande nel tempo (Figura 1B), mentre si possono verificare aggregati cellulari non specifici durante coltura ce...

Watch this Scientific Journal Video about Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells at JoVE.com

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Un metodo è stato sviluppato per derivare direttamente le cellule staminali neurali umane da cellule progenitrici ematopoietiche arricchite da cellule del sangue periferico.

Induzione diretta di cellule staminali neurali umane da cellule periferiche del sangue emopoietiche progenitrici

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

direct-induction-human-neural-stem-cells-from-peripheral-blood

Research

JoVE Journal

Developmental Biology

12.4K Views.  National Institutes of Health.  Un metodo è stato sviluppato per derivare direttamente le cellule staminali neurali umane da cellule progenitrici ematopoietiche arricchite da cellule del sangue periferico. 

Video: Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivazione di cellule cardiache progenitrici di cellule staminali embrionali

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Ricerca

Biologia dello sviluppo

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Induzione diretta di Hemogenic endotelio e Sangue da sovraespressione di fattori di trascrizione in cellule staminali pluripotenti umane

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolamento di cellule endoteliali progenitrici dal sangue del cordone ombelicale umano

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Cella singola fotoconversione in Zebrafish intatto vivente

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...