Gli autori desiderano ringraziare Soon-Cheol Hong (MD, Ph.D., Professore Associato, Dipartimento di Ostetricia e Ginecologia, Medical College Korea University) per la fornitura di tessuto placentare. Questo lavoro è stato sostenuto in parte da sovvenzioni (R1211902) dal National Research Foundation di Corea (NRF), Repubblica di Corea.

Etica dichiarazione: Lo studio è stato condotto placenta umana prospetticamente, con l&#39;approvazione del Institutional Review Board per la ricerca umana della University Korea (AN09085-001). Tutti gli esperimenti sono stati eseguiti in un Germ-Free Clean facility in camera presso il Medical Center Korea University. Il design e le procedure che utilizzano hPSCs sperimentale sono state approvate dal Institutional Review Board della Korea University Medical Center (AN12277-003). 1. Preparazione degli strumenti, terreni di coltura, e piatti <ol><li> Coat tutti i piatti della cultura con il 0,1% di gelatina. Salino Autoclave tampone fosfato (PBS) e pinze. Sterilizzare forbici chirurgiche, tubi microcentrifuga, e garze a 121 ° C sotto £ 15 psi per più di 15 min. </li><li> 0,25% tripsina-etilene ammina tetra-acetico preparati pre-riscaldato (tripsina-EDTA) e mezzi di pre-riscaldato cultura filtrato (DMEM) contenente siero bovino fetale 20% (FBS), 100 U / ml di penicillina, e 100 g / ml streptomycin prima di iniziare le procedure. </li></ol> 2. Isolamento delle cellule da placenta umana <ol><li> Ottenere tessuto placentare, dopo il parto cesareo, da parte delle donne sane in gravidanza normale fase di aborto o terapeutico a 7-32 settimane di gestazione dopo aver ottenuto il consenso informato scritto. </li><li> Hanno lo specialista ostetrico placenta umana piastre coriali chirurgicamente separati (HPC) dalla placenta; incubare questi in 0,25% tripsina-EDTA a 37 ° C per 20 minuti, e poi lavare con 5 ml di PBS. </li><li> Isolare villi coriali da HPC con le forbici; Tritare i villi e lavarli tre volte con 1 ml di PBS. Dopo macinazione, posizionare i villi coriali in una provetta per microcentrifuga con un puntale; lavare i campioni con 500 microlitri di PBS pipettando su e giù, e centrifugare le provette per 120 xg per 3 min. Lavare tessuti due volte con 500 ml di terreno di coltura pre-riscaldata. </li><li> Cellule culturali in media su piastre di gelatina rivestite a 37 ° C, in 5% di CO 2 </sub&gt;, e sotto il 95% di umidità. Pre-coat tutti i piatti con lo 0,1% di gelatina per 1 ora a 37 ° C. Le colonie di cellule fibroblasto-simili formeranno e crescita aderente circa 5-7 giorni dopo l&#39;inizio cultura nello stesso mezzo. </li><li> Sostituire il mezzo ogni 2-3 giorni fino al terzo passaggio. Durante la coltura, rimuovendo detriti cellulari galleggianti nel mezzo di coltura; crescita fibroblasti placentari si attaccheranno alla piastra. Coltivare le cellule di fibroblasti, come derivati ​​dal HPC fino al 10 ° passaggio e poi li raccolgono per le culture hESC dopo tripsinizzazione e mitomicina-C (10 mg / ml) trattamento. </li><li> Prima di congelamento HPC come campioni azionari, utilizzare trascrizione inversa-polymerase chain reaction (RT-PCR) per confermare che HPC non sono contaminati da agenti patogeni che normalmente infettano la placenta, tra cui micoplasma (M. fermentans, M. hyorhinis, M. arginina, M . orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. neurolyticum, M. hyopneumoniae,e M. capricolum) e le specie batteriche, Ureaplasma urealyticum, utilizzando un kit di rilevamento micoplasma. <ol><li> Seguire le istruzioni del produttore per effettuare RT-PCR, utilizzare tutte primer e miscele fornite nel kit. Utilizzare il surnatante delle cellule che sono state coltivate per 36 giorni per la rilevazione micoplasma. Aggiungere 3 ml di questi campioni per le miscele PCR ed eseguire la procedura 1 st PCR per 35 cicli, ciascuno costituito da 94 ° C per 30 sec, 55 ° C per 2 min, e 72 ° C per 1 min. </li><li> Aggiungere con cautela 0,5 ml di 1 ° prodotto PCR alla miscela PCR ed eseguire la procedura 2 ° PCR per 30 cicli usando le stesse condizioni di prima. Analizzare i prodotti amplificati di 1% gel di agarosio; applicare 10 ml di ogni del 1 ° e 2 ° prodotti di PCR per i gel. </li></ol></li><li> Se HPC si trovano ad essere contaminati da agenti patogeni, eliminarliutilizzando una combinazione di antibiotici (BM-ciclina), seguendo le istruzioni del produttore. Aggiungere un nuovo terreno contenente 10 mg / ml di BM-ciclina 1 per 3 giorni, e poi trattare le cellule con 5 mg / ml BM-ciclina 2 per altri 4 giorni. Ripetere questo ciclo per due volte e quindi verificare la presenza di contaminazione da micoplasma di nuovo. </li></ol> 3. Cellule raccolta umana Placenta derivati ​​condizionata Media (hPCCM) <ol><li> Dopo aver escluso contaminazione, trattare le cellule attaccate (85-90% di confluenza, (1,5 × 10 7 cellule / fiasco) con mitomicina-C (10 ug / ml) per 2 ore e 30 minuti, e le cellule lavate tre volte con 10 ml PBS. Incubare le cellule con mezzo di pre-riscaldato senza mitomicina-C per 24 ore. </li><li> Un giorno dopo il trattamento, incubare cellule in 10 ml di mezzo (DMEM-F12 integrato con il 20% di knock-out siero sostituzione [KOSR], 0,1 mm β-mercaptoetanolo, 1% NEAA, e l&#39;1% di penicillina-streptomicina). Dopo 24 ore di incubazione, il raccolto CM in un tubo da 15 ml efiltrata del CM con un filtro siringa da 0,22 micron. </li><li> Aggiungere altri 10 ml di nuovi media e di incubazione. Raccogliere tutte surnatanti dalle culture ogni giorno per 1 settimana e congelare il medium raccolto a -80 ° C. Evitare ripetuti cicli di congelamento-scongelamento. </li></ol> 4. Cellule Cultura di staminali embrionali umane (hESC) e induzione di cellule staminali pluripotenti (iPSCs) <ol><li> Ottenere linee di cellule staminali embrionali umane, H1 e H9 cellule (elencati nel Registro NIH hESC con il nome di WA01 e WA09, rispettivamente) e di cellule staminali indotte pluripotenti-IPSC-1: iPS (prepuzio) -1 e IPSC-2 (IISH1i- BM) dal Research Institute WiCell (Madison, WI, USA). Le cellule devono essere scongelati e coltivate seguendo le istruzioni del produttore. </li><li> Per il gruppo di controllo, le cellule di coltura su piatti matrice di membrana basale rivestite in diffuso mezzo di coltura definito senza siero-libero-alimentazione e a 37 ° C e in 5% di CO 2. Inizialmente, sementi 80-100 ciuffi di hPSCs in 35 mm piatti. Crescere cellule in questi piatti fino a 70-80% di confluenza e di routine cellule passaggio per sub-coltura una volta ogni 5-6 giorni, con metodi meccanici o enzimatici (dispasi). Lavare le cellule due volte con terreno e li piastra in un rapporto di 1: 4. Sostituire il supporto con terreno fresco ogni giorno. </li><li> Per i gruppi sperimentali, le cellule di coltura in hPCCM sui piatti pre-rivestite con 0,1% di gelatina e di eseguire passaging di routine una volta ogni 5 giorni, sia meccanicamente o enzimatica. Cellule piastra a rapporti nella gamma di 1: 6-1: 10. hPSCs allegare alla gelatina entro 2 giorni dopo il trasferimento. Sostituire il supporto con terreno fresco tutti i giorni. </li></ol> 5. Caratterizzazione di cellule staminali pluripotenti umane NOTA: Caratterizzare hPSCs utilizzando diversi metodi, come la colorazione fosfatasi alcalina, immunocitochimica, quantitativa real-time PCR (qRT-PCR), e western blotting. <ol><li> Immunofluorescenza <ol><li> Per immunofluorescence di colorazione, hPSCs cultura e fissare le cellule nelle camere di scorrimento 8 pozzetti con il 4% (w / v) paraformaldeide per 10 minuti; poi, le cellule permeabilizzate con 0,1% (v / v) Triton-X100 per 15 min, e bloccare le cellule per 1 ora con 3% (v / v) di siero di cavallo normale in PBS contenente 0,1% (v / v) Tween- 20. Lavare PBS due volte per 5 minuti tra ogni passo. </li><li> Incubare le cellule con l&#39;anticorpo primario (ottobre-4, SSEA4, TRA-1-81, TRA-1-60, diluizione di 1: 1.000) O / N a 4 ° C e con l&#39;anticorpo secondario. </li><li> Prima del montaggio, incubare le cellule con 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) per 5 minuti al buio. Lavare le cellule rigorosamente tre volte per 5 minuti ciascuno e secche cellule completamente, nel buio. Conservare le cellule in mezzo di montaggio fluorescenza e osservarli al microscopio a fluorescenza. </li></ol></li><li> qRT-PCR <ol><li> Isolare RNA totale da cellule utilizzando un kit RNeasy mini secondo le specifiche del produttore e quantificare l&#39;RNA estratto utilizzando un Nano goccia spettrofotometro. </li&gt; <li> Sintetizzare cDNA aggiungendo 2 ug di RNA totale per un oligo contenente 20 microlitri miscela di reazione (dT) primer e Superscript II trascrittasi inversa; seguire le istruzioni del produttore in questa procedura. </li><li> Amplificare il cDNA sintetizzato con un sistema iQ Bio-Rad iCycler, utilizzando iQ SYBR Green qPCR Master Mix e le sequenze di primer descritti nella tabella di riferimento S1. Normalizzare i valori di soglia ciclo dei geni di interesse a quelli di deidrogenasi gliceraldeide-3-fosfato (GAPDH). </li></ol></li><li> La fosfatasi alcalina (AP) colorazione <ol><li> Rilevare l&#39;attività AP utilizzando il cellulare ES Caratterizzazione Kit, secondo il protocollo del produttore. </li><li> Il giorno 5, aspirare i media e fissare i hPSCs con paraformaldeide al 4% in PBS per 12 min. Non overfix; fissa cellule per più di 2 min comporterà l&#39;inattivazione di fosfatasi alcalina. Aspirare il fissativo e lavare con 1x Sciacquare tampone. Noni pozzetti si asciughino tra i passaggi. </li><li> Aggiungere soluzione colorante sufficiente a coprire ogni pozzetto (1 ml per pozzetto di un piatto da 35 mm). Incubare le piastre al buio a temperatura ambiente per 15 min. </li><li> Dopo l&#39;incubazione, lavare i piatti con 1x Sciacquare tampone. Celle di copertura con PBS 1x per prevenire l&#39;essiccazione ed esaminare e immagine le cellule colorate utilizzando un microscopio Olympus. </li></ol></li><li> Western Blot <ol><li> Eseguire l&#39;analisi western blot come descritto in precedenza in 9. In breve, determinare le concentrazioni di proteine ​​in lisati cellulari. Risolvere uguali quantità di proteine ​​su un gel elettroforesi solfato-poliacrilammide (SDS-PAGE) gel di sodio dodecil e trasferire le proteine ​​su un fluoruro di polivinilidene (PVDF) membrana. </li><li> Bloccare la macchia e la sonda è O / N con anticorpi primari diversi a 4 ° C; successivamente incubare la macchia con anticorpi secondari HRP-coniugati per 1 ora. Lavare la membrana per tre volte per 10 minuti ogni volta tra incubazione. Rileva segnali mediante un reagente ECL. </li&gt; </ol></li><li> Short Tandem Repeat (STR) Analisi <ol><li> hPSCs sono state raccolte dal controllo e gruppi sperimentali allo stesso passo. DNA genomico è stato estratto utilizzando un kit Micro DNA, secondo le istruzioni del produttore. </li><li> Amplificare il DNA genomico per 16 loci genetici diversi utilizzando il kit CF / STR PCR amplificazione, secondo le specifiche del produttore e elettroforesi capillare è stata effettuata utilizzando un analizzatore genetico. </li></ol></li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+supports+undifferentiated+human+embryonic+stem+cell+growth+without+conditioned+medium.">Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.</a> Stem Cells. 23 (3), 315-323 (2005).</li><li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. 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Gli autori indicano conflitti di interesse.

Questo modello è stato sviluppato per propagare successo hPSCs, pur mantenendo le loro caratteristiche, in condizioni di coltura privo di alimentazione umanizzati senza aggiunta di esogeni fattori di crescita ricombinanti come bFGF o insulina, consentendo la manipolazione di hPSCs in un microambiente umanizzato. Supplementazione esogena bFGF è comune e l&#39;applicazione di substrati come Matrigel o laminina è essenziale per la coltura senza alimentatore di hPSCs 14. Questo pro...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

Un vantaggio di questo metodo di propagazione HPSC è che utilizza componenti secreti dalle cellule umane. Un passo più ciritical nel protocollo è l&#39;isolamento e la coltura di cellule derivate da placenta umana. Ciò richiede ed accurata dissezione da una parte precisa di HPC piastra villi. La Figura 1 mostra la procedura per l&#39;isolamento delle cellule derivate placenta umana. Questo processo è semplice e conveniente per l&#39;isolamento delle cellule, e può essere facilmente eseguita entro ...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel cultura Modello pluripotenti staminali umane propagazione delle cellule su Gelatina in Media Placenta condizionata

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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Research

JoVE Journal

Developmental Biology

7.8K Views.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

Video: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Incapsulamento alginato di cellule staminali pluripotenti usando un ugello coassiale

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Ricerca

Biologia dello sviluppo

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Coltura umane pluripotenti e neurali cellule staminali in un sistema di coltura cellulare chiuso per base e preclinica di ricerca

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Rapid neuronale differenziazione delle cellule staminali pluripotenti indotte per misurare l&#39;attività di rete su array Micro-elettrodo

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

Utilizzando umani indotti dell'epatocita-come di derivati da cellule staminali pluripotenti per scoperta della droga

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Un modello di Mouse chimerici del fegato umano di ipercolesterolemia familiare facendo uso degli epatociti derivati da cellule staminali pluripotenti indotte

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.