Name: stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates
Uploaded: 2016-06-17T14:00:00
Description: Watch this Scientific Journal Video about Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates at JoVE.com

Questo lavoro è sostenuto da NUS Messa in concessione (R-397-000-192-133) e ETPL Fondo Gap (R-397-000-198-592). GS è uno studioso NUS di ricerca. Gli autori desiderano ringraziare il Dr. Jiangwa Xing per il suo supporto tecnico micropatterning cellulare.

NOTA: Questo protocollo descrive la fabbricazione di PDMS stencil con 1.000 micron modelli di taglio laser e micropatterning della linea hESC, H9 con lo stencil PDMS. 1. progettazione e fabbricazione di PDMS Stencil per micropatterning <ol><li> Progettare il foglio di stencil con fori passanti della geometria desiderata e le dimensioni (ad esempio, 1.000 micron cerchi) e la guarnizione stencil utilizzando il computer-aided software di progettazione 10. </li><li> Laser-t...

<ol>
	<li>Graf, T., Stadtfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+embryonic+and+adult+stem+cells.">Heterogeneity of embryonic and adult stem cells.</a> Cell Stem Cell. 3 (5), 480-483 (2008).</li><li>Nishikawa, S., Goldstein, R. A., Nierras, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+human+induced+pluripotent+stem+cells+for+research+and+therapy.">The promise of human induced pluripotent stem cells for research and therapy.</a> Nat Rev Mol Cell Biol. 9 (9), 725-729 (2008).</li><li>Guilak, F., Cohen, D. M., Estes, B. T., Gimble, J. M., Liedtke, W., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+stem+cell+fate+by+physical+interactions+with+the+extracellular+matrix.">Control of stem cell fate by physical interactions with the extracellular matrix.</a> Cell Stem Cell. 5 (1), 17-26 (2009).</li><li>Dalby, M. J., Gadegaard, N., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+nanotopography+and+integrin-matrix+interactions+to+influence+stem+cell+fate.">Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate.</a> Nat Mater. 13 (6), 558-569 (2014).</li><li>Joddar, B., Ito, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+niche+substrates+for+embryonic+and+induced+pluripotent+stem+cell+cultures.">Artificial niche substrates for embryonic and induced pluripotent stem cell cultures.</a> J Biotechnol. 168 (2), 218-228 (2013).</li><li>Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D., Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+recapitulate+early+embryonic+spatial+patterning+in+human+embryonic+stem+cells.">A method to recapitulate early embryonic spatial patterning in human embryonic stem cells.</a> Nat Methods. 11 (8), 847-854 (2014).</li><li>Peerani, R., 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=Niche-mediated+control+of+human+embryonic+stem+cell+self-renewal+and+differentiation.">Niche-mediated control of human embryonic stem cell self-renewal and differentiation.</a> EMBO J. 26 (22), 4744-4755 (2007).</li><li>Lee, L. H., Peerani, R., Ungrin, M., Joshi, C., Kumacheva, E., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+human+embryonic+stem+cells+dissects+the+mesoderm+and+endoderm+lineages.">Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages.</a> Stem Cell Res. 2 (2), 155-162 (2009).</li><li>Hwang, Y. S., Chung, B. G., Ortmann, D., Hattori, N., Moeller, H. C., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwell-mediated+control+of+embryoid+body+size+regulates+embryonic+stem+cell+fate+via+differential+expression+of+WNT5a+and+WNT11.">Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11.</a> Proc Natl Acad Sci U S A. 106 (40), 16978-16983 (2009).</li><li>Toh, Y. C., Xing, J., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+integrin+and+E-cadherin-mediated+adhesions+to+spatially+control+heterogeneity+in+human+pluripotent+stem+cell+differentiation.">Modulation of integrin and E-cadherin-mediated adhesions to spatially control heterogeneity in human pluripotent stem cell differentiation.</a> Biomaterials. 50, 87-97 (2015).</li><li>Xing, J., Toh, Y. C., Xu, S., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+human+teratogen+detection+by+geometrically+confined+cell+differentiation+and+migration.">A method for human teratogen detection by geometrically confined cell differentiation and migration.</a> Sci Rep. 5, 10038 (2015).</li><li>Falconnet, D., Csucs, G., Grandin, H. M., Textor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+engineering+approaches+to+micropattern+surfaces+for+cell-based+assays.">Surface engineering approaches to micropattern surfaces for cell-based assays.</a> Biomaterials. 27 (16), 3044-3063 (2006).</li><li>Bauwens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+human+embryonic+stem+cell+colony+and+aggregate+size+heterogeneity+influences+differentiation+trajectories.">Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li><li>Khademhosseini, 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=Co-culture+of+human+embryonic+stem+cells+with+murine+embryonic+fibroblasts+on+microwell-patterned+substrates.">Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates.</a> Biomaterials. 27 (36), 5968-5977 (2006).</li><li>Mohr, J. C., de Pablo, J. J., Palecek, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+microwell+culture+of+human+embryonic+stem+cells.">3-D microwell culture of human embryonic stem cells.</a> Biomaterials. 27 (36), 6032-6042 (2006).</li><li>Yao, R., 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=Hepatic+differentiation+of+human+embryonic+stem+cells+as+microscaled+multilayered+colonies+leading+to+enhanced+homogeneity+and+maturation.">Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation.</a> Small. 10 (21), 4311-4323 (2014).</li><li>Mei, 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=Combinatorial+development+of+biomaterials+for+clonal+growth+of+human+pluripotent+stem+cells.">Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells.</a> Nat Mater. 9 (9), 768-778 (2010).</li><li>Melkoumian, Z., 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=Synthetic+peptide-acrylate+surfaces+for+long-term+self-renewal+and+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells.</a> Nat Biotechnol. 28 (6), 606-610 (2010).</li><li>Evans, N. D., 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=Substrate+stiffness+affects+early+differentiation+events+in+embryonic+stem+cells.">Substrate stiffness affects early differentiation events in embryonic stem cells.</a> Eur Cell Mater. 18, 1-13 (2009).</li><li>Carter, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haptotactic+islands:+a+method+of+confining+single+cells+to+study+individual+cell+reactions+and+clone+formation.">Haptotactic islands: a method of confining single cells to study individual cell reactions and clone formation.</a> Exp Cell Res. 48 (1), 189-193 (1967).</li><li>Jimbo, Y., Robinson, H. P., Kawana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+intracellular+calcium+and+electrical+activity+from+patterned+neural+networks+in+culture.">Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture.</a> IEEE Trans Biomed Eng. 40 (8), 804-810 (1993).</li><li>Wright, D., 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=Reusable,+reversibly+sealable+parylene+membranes+for+cell+and+protein+patterning.">Reusable, reversibly sealable parylene membranes for cell and protein patterning.</a> J Biomed Mater Res. A. 85 (2), 530-538 (2008).</li><li>Jinno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+multilayer+parylene-C+stencils+for+the+generation+of+patterned+dynamic+co-cultures.">Microfabricated multilayer parylene-C stencils for the generation of patterned dynamic co-cultures.</a> J Biomed Mater Res A. 86 (1), 278-288 (2008).</li><li>Jackman, R. J., Duffy, D. C., Cherniavskaya, O., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+elastomeric+membranes+as+dry+resists+and+for+dry+lift-off.">Using elastomeric membranes as dry resists and for dry lift-off.</a> Langmuir. 15 (8), 2973-2984 (1999).</li><li>Folch, A., Jo, B. H., Hurtado, O., Beebe, D. J., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+elastomeric+stencils+for+micropatterning+cell+cultures.">Microfabricated elastomeric stencils for micropatterning cell cultures.</a> J Biomed Mater Res. 52 (2), 346-353 (2000).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7 (8), 1018-1028 (2007).</li><li>Choi, J. H., Lee, H., Jin, H. K., Bae, J. S., Kim, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+neural+stem+cells+and+Purkinje+neurons+using+a+polydimethylsiloxane+(PDMS)+stencil.">Micropatterning of neural stem cells and Purkinje neurons using a polydimethylsiloxane (PDMS) stencil.</a> Lab Chip. 12 (23), 5045-5050 (2012).</li><li>Li, W., 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=NeuroArray:+a+universal+interface+for+patterning+and+interrogating+neural+circuitry+with+single+cell+resolution.">NeuroArray: a universal interface for patterning and interrogating neural circuitry with single cell resolution.</a> Sci Rep. 4, 4784 (2014).</li><li>Guvanasen, G. S., Mancini, M. L., Calhoun, W. A., Rajaraman, S., DeWeerth, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polydimethylsiloxane+Microstencils+Molded+on+3-D-Printed+Templates.">Polydimethylsiloxane Microstencils Molded on 3-D-Printed Templates.</a> J Microelectromech S. 23 (5), 1045-1053 (2014).</li><li>Palchesko, R. N., Zhang, L., Sun, Y., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+polydimethylsiloxane+substrates+with+tunable+elastic+modulus+to+study+cell+mechanobiology+in+muscle+and+nerve.">Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.</a> PLoS One. 7 (12), 51499 (2012).</li><li>Chowdhury, F., 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=Material+properties+of+the+cell+dictate+stress-induced+spreading+and+differentiation+in+embryonic+stem+cells.">Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells.</a> Nat Mater. 9 (1), 82-88 (2010).</li><li>Gjorevski, N., Boghaert, E., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Epithelial-Mesenchymal+Transition+by+Transmission+of+Mechanical+Stress+through+Epithelial+Tissues.">Regulation of Epithelial-Mesenchymal Transition by Transmission of Mechanical Stress through Epithelial Tissues.</a> Cancer Microenviron. 5 (1), 29-38 (2012).</li><li>Thery, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+as+a+tool+to+decipher+cell+morphogenesis+and+functions.">Micropatterning as a tool to decipher cell morphogenesis and functions.</a> J Cell Sci. 123, 4201-4213 (2010).</li><li>Eroshenko, N., Ramachandran, R., Yadavalli, V. K., Rao, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+substrate+stiffness+on+early+human+embryonic+stem+cell+differentiation.">Effect of substrate stiffness on early human embryonic stem cell differentiation.</a> J Biol Eng. 7 (1), 7 (2013).</li></ol>

Fabbricazione di stencil micropatterning micropatterning Stencil fornisce un metodo ideale per generare microdisegni HPSC per indagare nicchia mediata differenziazione patterning. Il vantaggio principale di stencil patterning rispetto ad altre tecniche micropatterning, come la stampa microcontact e photopatterning, è che non richiede la modifica della superficie e può essere implementato su supporti TCP convenzionali. Pertanto, terreni di coltura ottimizzati e riv...

Le cellule staminali pluripotenti umane (hPSCs), comprese le cellule staminali embrionali (hESC) e le cellule staminali pluripotenti indotte (hiPSCs), sono ampiamente sfruttati in medicina rigenerativa, nonché la modellazione sperimentale di organogenesi normale e malati a causa del loro potenziale di differenziazione in linee cellulari di tutti tre strati germinali 1,2. I destini di differenziazione di hPSCs sono estremamente sensibili ai fattori ambientali locali in grado di modulare i processi meccanotrasduzione autocrini o paracrini segnalazione 1 e mediate da stimoli fisici 3-5. Cellulare micropatterning comprende un insieme di tecniche che sono state sviluppate per organizzare spazialmente la geometria e la posizione di una popolazione di cellule come un mezzo per controllare il microambiente cellulare locale, come le interazioni cellula-cellula 6 e interazioni cellula-matrice 3. Nel contesto di hPSCs, micropatterning cellulare è stato impiegato per ottenere informazioni importanti sul modo in cui nicchia dipendeent segnalazione autocrina modula hESC pluripotenza decisioni-differenziazione 7 e organizzazione in primi modelli di differenziazione embrionali 6. 2D e 3D hPSCs micropatterned sono stati utilizzati per controllare la dimensione delle colonie di modelli multicellulari, che a sua volta influenzato decisioni differenziazione nei tre strati germinali 8,9. Abbiamo impiegato multicellulari microdisegni HPSC di modulare la portata di cellula-cellula e cellula-matrice interazioni all&#39;interno di una colonia HPSC per sondare come integrina-E-caderina crosstalk può dare origine a destino cellulare eterogeneità 10. Le manifestazioni delle suddette relazioni aprire nuove strade verso l&#39;applicazione di microdisegni multicellulari di hPSCs come modelli sperimentali per lo screening tossicità dei farmaci per le malattie dello sviluppo 11, per studiare l&#39;effetto di fattori di crescita e ormoni durante tessuti o lo sviluppo degli organi, e per svelare la formazione di modelli tessuti. Una miriade di cellule micropattertecniche ning sono stati sviluppati come recensito da Falconnet et. al. 12, ma solo una manciata, come il micro-contatto stampa 7,8,13, cultura micropozzetti 14,15, photopatterning 6 e microstencils 16 sono state implementate con successo con hPSCs. La sfida con hPSCs micropatterning risiede nella loro vulnerabilità e un requisito stringente di matrici specifiche extracellulare (ECM) e condizioni di crescita per il fissaggio e la sopravvivenza cellulare. Per i modelli 2D HPSC, stampa micro-contatto è uno dei metodi più comuni per generare microdisegni HPSC per la cultura dei tessuti e del vetro substrati 13. Il metodo può essere utilizzato per modello ECM comune utilizzato in coltura HPSC, compresi laminina e membrana basale matrici, come Matrigel. Tuttavia, esso richiede in genere un processo di rivestimento in due fasi aiutati da poli-D-lisina, e ha bisogno di particolari condizioni atmosferiche e di umidità inerti per rendere microdisegni ECM stabili per hPSCs di allegare a 6,13. La più importante considerazione di ciascun metodo micropatterning è se il regime di modifica della superficie in grado di generare modelli di ECM HPSC-adesivo alla risoluzione geometrica desiderata, riducendo al minimo l&#39;adesione cellulare non specifica alle aree circostanti. Qui, si segnala l&#39;uso di stencil micropatterning come un metodo semplice per generare microdisegni HPSC senza ulteriori passaggi la modifica della superficie prima della generazione di modelli ECM adesivi per hPSCs per fissare su. La cella comprende stencil di una sottile membrana, foglio per esempio, polidimetilsilossano (PDMS), con Micron per millimetriche dimensioni fori passanti sigillati su un substrato di coltura cellulare per contenere fisicamente rivestimenti ECM e hPSCs successivamente seminate. Come stencil patterning agisce trattenendo fisicamente la posizione in cui HPSC può accedere e collegare direttamente al substrato sottostante ECM rivestite, questo metodo è compatibile con vari substrati che possono sostenere culture HPSC. Gli unici requirement è che la scelta del materiale matrice può formare una tenuta reversibile con il substrato. Tali substrati comprendono convenzionale polistirene coltura tissutale (TCPS) 17, substrati coniugati ligando 18, così come substrati elastomerici rigidità sintonizzabile (ad es., PDMS) 19. Questo metodo permette anche di rivestimento di diversi ECM, come ad esempio vitronectina (o proteine ​​VTN), laminina e matrici membrana basale (ad esempio, Matrigel e Geltrax) per consentire il corretto fissaggio e la differenziazione di hPSCs. Pertanto, siamo in grado di trasferire ottimizzati configurazioni ECM-substrato per una specifica linea di HPSC a stencil micropatterning per ottimale cellula-matrice di adesione, la sopravvivenza e la differenziazione. Recentemente, un metodo simile è stato anche segnalato per dirigere la differenziazione epatica da hESC micropatterning utilizzando poli (metacrilato di metile) (PMMA) matrici di micro-stencil 16. stencil cellulari possono essere fabbricate con materiali diversi, tra cui incontratials 20,21, poli (p-xililene) polimeri 22,23, PMMA 16 e più comunemente, PDMS 24-28. Silicone e poli (p-xililenici) polimeri stencil richiedono incisione diretta dei fori passanti con attrezzature specializzate 20-23, che limita la loro accessibilità agli utenti biologici. PDMS stencil possono essere fabbricati con metodi diversi a seconda della dimensione del tratto richiesto, che varia tipicamente da 3 micron a 2000 micron 11,26-29. Se piccole caratteristiche si desiderano, fogli Stenciling sottili possono essere prodotti da stampa stampaggio PDMS pre-polimero su un modello di silicio microfabbricazione contenente rilievi dei microdisegni 28. Per le caratteristiche&gt; 1.000 micron, un taglio laser CO 2 fornisce un metodo semplice e di basso costo per tagliare direttamente i modelli su un foglio pre-fuso PDMS durante stencil fabbricazione. La riciclabilità dei stencil PDMS li rende anche conveniente per condurre una serie di esperimenti con sufficiente coerenza. <p class = &quot;jove_content&quot;&gt; Qui, vi presentiamo la metodologia dettagliata per la fabbricazione di uno stencil PDMS con 1.000 micron caratteristiche da taglio laser e la generazione di microdisegni hESC. Questi microdisegni hESC sono stati usati per modulare l&#39;entità della integrina ed E-caderina mediati aderenze all&#39;interno di una colonia hESC coesa in modo da indagare come la polarizzazione spaziale di adesione cellulare portato nella cella destino eterogeneità 10.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;2 mm thick PDMS sheet</td>
			<td style="width:191px;">Specialty Silicone Products Inc., USA</td>
			<td style="width:119px;">SSPM823-.005</td>
			<td style="width:423px;">Used to form reservoir for stencil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">120-150 &#956;m thick PDMS sheet</td>
			<td style="width:191px;">Specialty Silicone Products Inc., USA</td>
			<td style="width:119px;">SSPM823-.040</td>
			<td style="width:423px;">Used to form stencil&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">60 mm petri dish</td>
			<td style="width:191px;">&#160;Nunc Nunclon Delta</td>
			<td style="width:119px;">150326</td>
			<td style="width:423px;">Substrate for micropatterning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase</td>
			<td style="width:191px;">Accutase, Merck Millipore, Singapore</td>
			<td style="width:119px;">SCR005</td>
			<td style="width:423px;">Enzyme to break H9 Cells into single cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Activin&#160;&#160;</td>
			<td style="width:191px;">R&#38;D Systems, Singapore</td>
			<td style="width:119px;">338-AC-010</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BMP4&#160;</td>
			<td style="width:191px;">R&#38;D Systems, Singapore</td>
			<td style="width:119px;">338-BP-010</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasma system&#160;</td>
			<td style="width:191px;">&#160;Femto Science, Korea</td>
			<td style="width:119px;">CUTE-MP</td>
			<td style="width:423px;">For plasma oxidation of stencil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dispase</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">7923</td>
			<td style="width:423px;">Enzyme used to weaken the cell-ECM adhesion during passaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:191px;">GIBCO, USA</td>
			<td style="width:119px;">11330032</td>
			<td style="width:423px;">Basal medium for H9 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF2</td>
			<td style="width:191px;">&#160;R&#38;D Systems, Singapore</td>
			<td style="width:119px;">233&#8211;FB&#8211;025</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">H9 Cell line</td>
			<td style="width:191px;">WiCell Research Institute, Inc., USA</td>
			<td style="width:119px;">WA09</td>
			<td style="width:423px;">Human embryonic stem cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">hESC-qualified basement membrane matrix</td>
			<td style="width:191px;">Matrigel, BD Biosciences, Singapore</td>
			<td style="width:119px;">354277</td>
			<td style="width:423px;">Extra-cellular matrix coating to support growth of H9 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope</td>
			<td style="width:191px;">Leica Microsystems, Singapore</td>
			<td style="width:119px;">DMi1</td>
			<td style="width:423px;">For capturing bright-field images</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser cutter</td>
			<td style="width:191px;">Epilog Helix 24 Laser System</td>
			<td style="width:119px;"></td>
			<td style="width:423px;">Used to generate through holes in PDMS sheet</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">mTeSR&#8482;1 medium&#160;</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">5850</td>
			<td style="width:423px;">Maintainence medium for H9 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PDMS&#160;</td>
			<td style="width:191px;">SYLGARD&#174; 184, Dow Corning Co., USA</td>
			<td style="width:119px;">3097358-1004</td>
			<td style="width:423px;">Used for sticking the PDMS stencil and reservior</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ROCKi Y27632</td>
			<td style="width:191px;">Calbiochem, Merck Millipore, Singapore</td>
			<td style="width:119px;">688000</td>
			<td style="width:423px;">Maintains H9 cells as single cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">STEMdiff&#8482; APEL&#8482; medium&#160;</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">5210</td>
			<td style="width:423px;">Differentiation medium for H9 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene terephthalate film</td>
			<td style="width:191px;">SureMark Singapore</td>
			<td style="width:119px;">SQ-6633</td>
			<td style="width:423px;">Used to form stencil&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture compatible non-ionic surfactant</td>
			<td style="width:191px;">Pluronic acid F-127, Sigma, Singapore</td>
			<td style="width:119px;">P2443</td>
			<td style="width:423px;">Passivating reagent to repel cell adhesion in non-micropatterned substrates</td>
		</tr>
	</tbody>
</table>

stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

In questo articolo, descriviamo la fabbricazione di uno stencil cella utilizzando una taglierina laser per generare 1.000 micron caratteristiche. La matrice è composta da 2 parti: un sottile foglio stencil (circa 100-200 micron di spessore), contenente la micropattern fori passanti, ed una guarnizione PDMS a contenere la soluzione di rivestimento ECM o sospensione cellulare. Qui, 127 micron e 2 mm di spessore fogli PDMS disponibili in commercio sono stati usati come foglio stencil e la ...

Watch this Scientific Journal Video about Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates at JoVE.com

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Le cellule staminali pluripotenti umane (hPSCs) hanno la capacità intrinseca di differenziare e di auto-organizzarsi in schemi dei tessuti distinti; anche se questo richiede la presentazione di gradienti spaziali ambientali. Presentiamo stencil micropatterning come metodo semplice e robusto per generare gradienti biochimici e meccanici per controllare modelli differenziazione HPSC.

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

The overall goal of this stencil-based micropatterning method, for human pluripotent stem cells, is to provide a simple and robust means to generate spatial environmental gradients that control stem cell differentiation patterns. This method can help you answer key questions in tissue patterning using a human stem cell model, such as how spatial gradients in cell-cell and cell matrix adhesion forces can help to organize stem cell differentiation fates. The advantage of this method is that it does not require additional surface modification to generate cell adhesive ECM patterns for cells to attach to.This means the method is compatible with various ECM and substrate configurations, specific for each cell line. Visual demonstration of this method is critical, as the micropatterning steps are difficult to learn. Because it requires precise and careful handling of the stencil and the cells.For this procedure, the PTMS stencils must already be designed and fabricated. Likewise, have human EFC cells growing on basement membrane matrix, such that the colonies will be ready for seeding later in the procedure. To begin, load a dish with 700 microliters of 70%analytical-grade ethanol, in ultra-pure water.Then, place the PDMS stencil on the solution, and set the dish into a bio-safety cabinet to dry out overnight. The next day, check that no bubbles have formed under the stencil. Then, field the dish until the cells can be seeded onto the stencil.Prepare an aliquot of human ESC-quality basement membrane matrix by adding 16.7 milliliters of DMF12, to make a 1.5x solution and keep it on ice. Treat the substrate with 100 watts of oxygen plasma for 90 seconds. Don't open the dish until it is inside the plasma chamber, and quickly cover it up after the treatment.This treatment will prevent bubbles from forming when the substrate is immersed in solution. Now, cover the stencil with 450 microliters of prepared 1.5x basement membrane matrix solution. Then, seal the dish with parafilm and incubate it for five hours.Before proceeding, check over the six well plates of prepared HESC colonies. Identify and aspirate off colonies of differentiated cells that have lost their typical HESC morphology. They should all appear rounded, have a tightly-packed epithelial morphology, and have a high nucleus-to-cytoplasm ratio with prominent nucleoli.Then, wash each well of plated cells with two milliliters of DMF12 two times. After the second wash, add one milliliter of digestive enzymes to each well, such as accutase and incubate the plate for eight minutes at 37 degrees Celsius. After the incubation, tap the plate gently to detach all the colonies from the substrate.Next, rinse each well with at least four milliliters of media for every milliliter of digestive enzyme applied to the well, and collect the cell suspensions into a single 15 milliliter conical tube. Centrifuge the cells at 200 g for three minutes at room temperature to form a cell pellet. Then aspirate the supernatant, and re-suspend the cells in 400 microliters of HESC maintenance media, supplemented with 10 micromolar of rock inhibitor.Triturate the cells gently three times to break up the clumps. Next, correct the suspensions'density. Mix it well, and make a 200 microliter 1:20 dilution in media.Count the cells in the dilution using a hemocytometer. Then, dilute the main suspension as needed, using media with rock inhibitor. To proceed, add the required number of cells onto each prepared stencil.Only a mono-layer of cells should be deposited, so spread them out evenly. Let the stencil sit undisturbed for five minutes, so the cells can settle. Then, carefully transfer the loaded stencils to a incubator, keeping the dishes level throughout the transfer.Let the cells attach to the stencils over an hour-long incubation. After the cells have been allowed to attach, examine them under a microscope to ensure that they did so properly. Transfer the stencil back to the hood, and aspirate the unattached cells off the stencil.Next, add two milliliters of 0.5%compatible non-ionic surfactant in DMF12 to the area surrounding the stencil. Swirl the solution around to cover the stencil. Then, use a pair of autoclaved forceps to gently peel the stencil off the dish.After removing the stencil, inspect the micropatterend cells under a microscope. The non-ionic surfactant prevents these cells from drying out. Let the micropatterend cells incubate in the surfactant-supplemented media solution for 10 minutes at 37 degrees Celsius.After 10 minutes, aspirate away the solution, and wash the plate with two milliliters of normal DMF12 medium, three times. After the washes, add two milliliters of HESC maintenance medium with rock inhibitor. Once again, inspect the cells to check that the micropatterns have formed properly.Then, incubate the cells at 37 degrees Celsius overnight. The next day, aspirate the medium, wash the cells once with DMF12, and then, add two milliliters of supplemented differentiation medium, to induce the cells to undergo mesoendoderm differentiation. Later, analyze the results.The fabrication of the cell stencil using a laser cutter, provided 1000 micron features. The stencil was composed of a thin stenciling sheet with micropatterned through holes, and a PDMS gasket used to contain the cells. Using this tool, micropatterened HESCs were investigated during mesoendoderm differentiation.H9-HESC colony peripheries have higher integrin adhesions than cells at the colony interior. Thus, the cells differentiate into brachiary T-positive mesoendoderm cells, when induced with activan, BMP4, and FGF2. When such cells were patterned into monolayers of different geometries, geometric and isotropiate right angles and at convex curvatures, led to a local concentration of integrin mediated traction forces, and resulted in increased mesoendoderm differentiation.T expression intensity at different localities within a single colony showed that the extent of meso endoderm induction was indeed higher in sharp corners of square and rectangular colonies, and at the convex curvatures of a semi-circular arc. This demonstrates that cell adhesion mediated mechanical forces can be modulated using the described method. Before attempting the procedure, it is important to optimize the matrix coating time, the cell digestion and attachment time, for the specific human pluripotent stem cell line that you are working with.

Research

JoVE Journal

Developmental Biology

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

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 ...

Ricerca

Biologia dello sviluppo

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Aggregate ottimizzazione Dimensione in pozzetti per sospensione a base cardiaco differenziazione delle cellule staminali umane pluripotenti

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

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

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, ...

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

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 ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Differenziazione efficiente delle pluripotenti cellule staminali per NKX6-1<sup&gt; +</sup&gt; progenitori pancreatici

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

La differenziazione in vitro di pluripotenti umane Cellule Staminali in cellule trofoblastiche

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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

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 ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

Mappatura dell'Emergente Organizzazione Spaziale delle Cellule Mammarie utilizzando Micropatterns e Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...