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

Dette arbeidet støttes av NUS Oppstart stipend (R-397-000-192-133) og ETPL Gap fondet (R-397-000-198-592). GS er en NUS Forskning lærd. Forfatterne ønsker å takke dr Jiangwa Xing for henne teknisk support på celle micropatterning.

MERK: Denne protokollen beskriver fabrikasjon av PDMS sjablong med 1000 mikrometer mønstre av laserskjæring og micropatterning av hESC linje, H9 bruke PDMS sjablong. 1. Design og fabrikasjon av PDMS Stencil for Micropatterning <ol><li> Design spraye arket med gjennomgående hull med ønsket geometri og størrelse (f.eks 1000 mikrometer sirkler) og sjablongen pakning bruker dataassistert design software 10. </li><li> Laser-kutt spraye arket og pakning på 120-150 nm og...

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Fabrikasjon av micropatterning sjablonger Stencil micropatterning gir en ideell metode for å generere hPSC micropatterns for å undersøke nisje-mediert differensiering mønster. Den viktigste fordelen med sjablongen mønster i forhold til andre micropatterning teknikker, slik som mikrokontakttrykking og photopatterning, er at den ikke krever overflatemodifisering og kan implementeres på konvensjonelle TCPS substrater. Derfor kan optimaliserte dyrkningsmedier og E...

Humane pluripotente stamceller (hPSCs), inkludert embryonale stamceller (hESCs) og induserte pluripotente stamceller (hiPSCs), allment utnyttes i regenerativ medisin samt eksperimentelle modellering av normal og sykelig organogenese på grunn av deres differensiering potensial inn i cellen linjer av alle tre bakterie lag 1,2. Differensieringen skjebne hPSCs er svært følsomme for lokale miljøfaktorer som kan modulerer autokrine eller parakrine signaliserer en samt mechanotransduction prosesser mediert av fysiske signaler 3-5. Cell micropatterning omfatter et sett av teknikker som er utviklet for å romlig organisere geometri og plassering av en cellepopulasjon som et middel for å kontrollere den lokale mobilmikromiljøet, slik som celle-celle interaksjoner 6 og celle-matriks interaksjoner 3. I sammenheng med hPSCs har celle micropatterning vært ansatt for å oppnå betydelige innsikt i hvordan nisje-avhengigeent autokrint signale modulerer hESC pluripotency-differensiering beslutninger 7 og organisasjon i tidlig embryonale differensieringsmønster 6. 2D og 3D micropatterned hPSCs har blitt brukt til å styre kolonien størrelsen på flercellede mønstre, som i sin tur påvirket differensiering beslutninger i de tre bakterie lag 8,9. Vi har anvendt flercellede hPSC micropatterns å modulere den grad av celle-celle og celle-matriks-vekselvirkninger innenfor et hPSC koloni å undersøke hvordan integrin-E-cadherin krysstale kan gi opphav til celle skjebne heterogenitet 10. Demonstrasjonene fra de ovennevnte rapporter åpne nye veier mot anvendelsen av flercellede micropatterns av hPSCs som eksperimentelle modeller for narkotika toksisitet screening for utviklings sykdommer 11, for å studere effekten av vekstfaktorer og hormoner i løpet av vev eller organ utvikling, og å rakne dannelsen av vev mønstre. En myriade av celle micropatterNing teknikker har blitt utviklet som anmeldt av Falconnet et. al. 12, men bare en håndfull, for eksempel mikro-kontakt utskrift 7,8,13, mikrokultur 14,15, photopatterning 6 og microstencils 16 har blitt implementert med hPSCs. Utfordringen med micropatterning hPSCs ligger i deres sårbarhet og strenge krav om konkrete ekstracellulære matriser (ECM) og vekstvilkår for cellebinding og overlevelse. For 2D hPSC mønstre, er mikro-kontakt utskrift en av de vanligste metodene for å generere hPSC micropatterns på vev kultur og glass underlag 13. Fremgangsmåten kan brukes til å påføre mønster felles ECM som brukes i hPSC kultur, inkludert laminin og basalmembran matriser, slik som Matrigel. Imidlertid, er det vanligvis krever en totrinns belegningsprosess hjulpet av poly-D-lysin, og trenger spesielle inerte atmosfæriske og fuktighetsforhold for å lage stabile ECM micropatterns for hPSCs å feste på 6,13. Den fremste hensyn til hver micropatterning metode er om overflaten ombygging regime kan generere hPSC-lim ECM mønstre på ønsket geometrisk oppløsning mens uspesifikke cellebinding minimere til de omkringliggende områdene. Her rapporterer vi bruk av sjablongen micropatterning som en enkel metode for å generere hPSC micropatterns uten ytterligere overflatemodifikasjonstrinn før generering av selvklebende ECM mønstre for hPSCs å feste på. Cellen sjablongen består av en tynn membran, for eksempel polydimetylsiloksan (PDMS) ark, med mikron til millimeter størrelse, gjennomgående hull forseglet på en cellekultur substrat for fysisk å inneholde ECM belegg og deretter sådd hPSCs. Som sjablong mønster virker ved fysisk tilbakeholdende det sted hvor hPSC kan få tilgang til og festes direkte på den underliggende ECM belagte substrat, er denne metode som er kompatibel med forskjellige substrater som kan støtte hPSC kulturer. De eneste requirement er at valg av sjablongen materiale kan danne en reversibel tetning med substratet. Disse substrater inkluderer konvensjonelle vevskulturpolystyren (TCPS) 17, ligand-konjugerte substrater 18, samt elastomere substrater med avstembar stivhet (f.eks., PDMS) 19. Denne metoden gir også belegg av forskjellig ECM, som vitronectin (eller VTN protein), laminin og basalmembran matriser (f.eks Matrigel og Geltrax) for å få riktig feste og differensiering av hPSCs. Derfor kan vi overføre optimalisert ECM-substrat konfigurasjoner for en bestemt hPSC linje til sjablong micropatterning for optimal celle-matrix adhesjon, overlevelse og differensiering. Nylig har en lignende metode også blitt rapportert til å dirigere hepatisk differensiering av micropatterning hESCs ved hjelp av poly (metylmetakrylat) (PMMA) mikro sjablong matriser 16. Celle sjablonger kan fremstilles av forskjellige materialer, inkludert møttals 20,21, poly (p-xylylen) polymerer 22,23, PMMA 16 og mest, PDMS 24-28. Silisium og poly (p-xylylen) polymerer sjabloner krever direkte etsning av de gjennomgående hullene med spesialutstyr 20-23, noe som begrenser deres biologiske tilgjengelighet til brukerne. PDMS sjabloner kan fremstilles ved forskjellige metoder, avhengig av trekkstørrelse nødvendig, noe som er typisk i området fra 3 mikrometer til 2000 mikrometer 11,26-29. Hvis små funksjoner er ønskelig, kan tynne spraye plater produseres ved trykk molding PDMS pre-polymer på en microfabricated silisium mal som inneholder relieffer av de micropatterns 28. For funksjoner&gt; 1000 mikrometer, gir et CO 2 laser cutter en enkel og rimelig metode for å direkte kutte mønstrene på en pre-støpt PDMS ark under sjablongen fabrikasjon. Den resirkulering av PDMS sjablonger gjør dem også kostnadseffektivt å gjennomføre en rekke eksperimenter med tilstrekkelig konsistens. <p class = &quot;jove_content&quot;&gt; Her presenterer vi detaljert metodikk for fabrikasjon av en PDMS sjablong med 1000 mikrometer funksjoner ved laserskjæring og generering av hESC micropatterns. Disse hESC micropatterns ble anvendt for å modulere den grad av integrin og E-cadherin mediert adhesjon innenfor en sammenhengende hESC koloni for derved å undersøke hvordan romlig polarisering av celleadhesjon resulterte i celle skjebne heterogenitet 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.

I denne artikkelen beskriver vi fabrikasjon av en celle sjablong ved hjelp av en laser cutter å generere 1000 mikrometer funksjoner. Sjablongen var sammensatt av 2 deler: en tynn spraye ark (ca. 100-200 mikrometer tykk) inneholdende micropattern gjennomgående hull, og et PDMS pakning for å inneholde den ECM beleggløsningen eller cellesuspensjon. Her ble 127 mikrometer og 2 mm tykke kommersielt tilgjengelige PDMS ark anvendes som spraye ark og pakning henholdsvis. Andre fremgangsmåte...

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

Menneskelige pluripotente stamceller (hPSCs) har den iboende evne til å differensiere og selvtillit organisere inn i forskjellige vev mønstre; selv om dette krever presentasjon av romlige miljø gradienter. Vi presenterer sjablong micropatterning som en enkel og robust metode for å generere biokjemiske og mekaniske gradienter for å styre hPSC differensieringsmønster.

Stencil Micropatterning of Human Pluripotent stamceller for undersøkelser romlige organiseringen av differensiering Fates

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

Research

JoVE Journal

Developmental Biology

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

Storskala produksjon av cardiomyocytes fra Menneskelig Pluripotent stamceller ved hjelp av en meget reproduserbar lite molekyl-Based Differensiering Protocol

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

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

Aggregate Størrelse Optimalisering i mikro for Suspension basert Cardiac Differensiering av menneskelige Pluripotent stamceller

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 neuronal Differensiering av Induced Pluripotent stamceller for måling Network Aktivitet på Micro-elektrode Arrays

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

Rettet differensiering av Primitive og Definitive blodkreft Progenitors fra menneskelige Pluripotent stamceller

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

Effektiv Differensiering av pluripotent stamceller til å NKX6-1<sup&gt; +</sup&gt; bukspyttkjertelen stamfedre

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

In Vitro Differensiering av menneskelige pluripotent stamceller til trophoblastic Cells

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

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent stamceller

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

Effektiv differensiering av Human Pluripotent stamceller i leverceller

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

I Vitro generasjon av Somite derivater fra menneske indusert stamceller Pluripotent

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

Kartlegging av emergent romlig organisering av pattedyrceller bruker Micropatterns og kvantitativ 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 ...