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 arbejde understøttes af NUS Start tilskud (R-397-000-192-133) og ETPL Gap Fund (R-397-000-198-592). GS er en NUS Research lærd. Forfattere vil gerne takke Dr. Jiangwa Xing for hendes teknisk support på celle micropatterning.

BEMÆRK: Denne protokol beskriver fremstillingen af ​​PDMS stencil med 1.000 um mønstre ved laserskæring og micropatterning af hESC linje, H9 hjælp af PDMS stencil. 1. Design og Fremstilling af PDMS Stencil for Micropatterning <ol><li> Design af stenciling ark med gennemgående huller i den ønskede geometri og størrelse (f.eks, 1.000 um cirkler) og stencil pakning ved hjælp af computer-aided design software 10. </li><li> Laser-cut stenciling plader og pakning ...

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Fabrikation af micropatterning stencils Stencil micropatterning giver en ideel metode til at generere hPSC micropatterns til undersøgelse niche-medieret differentiering mønster. Den vigtigste fordel ved stencil mønster i forhold til andre micropatterning teknikker, såsom microcontact udskrivning og photopatterning, er, at det ikke kræver overfladebehandling og kan implementeres på konventionelle TCPS substrater. Derfor kan optimerede dyrkningsmedier og ECM bel...

Humane pluripotente stamceller (hPSCs), herunder embryonale stamceller (hESCs) og inducerede pluripotente stamceller (hiPSCs), er i vid udstrækning udnyttet i regenerativ medicin samt eksperimentel modellering af normale og syge organogenese på grund af deres differentiering potentiale i cellelinier af alle tre kim lag 1,2. Differentieringen skæbner hPSCs er meget følsomme over for de lokale miljømæssige faktorer, der kan modulere autokrine eller parakrine signalering 1 samt mechanotransduction processer medieret af fysiske signaler 3-5. Celle micropatterning omfatter et sæt af teknikker, der er udviklet til rumligt organiserer geometrien og placeringen af en cellepopulation som et middel til at styre den lokale cellulære mikromiljø, såsom celle-celle interaktioner 6 og celle-matrix-interaktioner 3. I forbindelse med hPSCs har celle micropatterning været ansat til at vinde betydelige indsigt i, hvordan niche-afhængigeent autokrine signalering modulerer hESC pluripotens-differentiering beslutninger 7 og organisation i tidlige embryonale differentiering mønstre 6. 2D og 3D mikromønstrede hPSCs er blevet anvendt til at styre koloni størrelsen af flercellede mønstre, som igen påvirkes beslutninger differentiering til de tre kimlag 8,9. Vi har ansat flercellede hPSC micropatterns at modulere omfanget af celle-celle- og celle-matrix-interaktioner inden for en hPSC koloni til at probe hvordan integrin-E-cadherin krydstale kan give anledning til celleskæbne heterogenitet 10. Demonstrationerne fra ovenstående rapporter åbne nye veje mod anvendelsen af flercellede micropatterns af hPSCs som eksperimentelle modeller for lægemiddeltoksicitet screening for udviklingsmæssige sygdomme 11, for at studere effekten af vækstfaktorer og hormoner under væv eller organ udvikling, og at optrævle dannelsen af væv mønstre. Et utal af celle micropatterning teknikker er blevet udviklet som gennemgået af Falconnet et. al. 12, men kun en håndfuld, såsom mikro-kontakt udskrivning 7,8,13, mikrobrønd kultur 14,15, photopatterning 6 og microstencils 16 er blevet gennemført med succes med hPSCs. Udfordringen med micropatterning hPSCs ligger i deres sårbarhed og en stringent krav om specifikke ekstracellulære matricer (ECM) og vækstbetingelser for cellebinding og overlevelse. For 2D hPSC mønstre, mikro-kontakt trykning er en af de mest almindelige metoder til at generere hPSC micropatterns på vævskultur og glas substrater 13. Fremgangsmåden kan anvendes til pattern fælles ECM anvendt i hPSC kultur, herunder laminin og basalmembran matricer, såsom Matrigel. Men kræver det typisk en to-trins coating proces hjulpet af Poly-D-lysin, og kræver særlige inaktive atmosfæriske og fugtighedsforhold at gøre stabile ECM micropatterns for hPSCs at vedhæfte på 6,13. Den forreste overvejelse af hver micropatterning metode er, om overfladen modifikation regime kan generere hPSC-klæbende ECM mønstre på den ønskede geometriske opløsning samtidig minimere uspecifik celle tilknytning til de omkringliggende områder. Her rapporterer vi brug af stencil micropatterning som en simpel metode til at generere hPSC micropatterns uden yderligere overfladebehandling skridt forud for generering af selvklæbende ECM mønstre for hPSCs at vedhæfte på. Cellen stencil består af en tynd membran, f.eks polydimethylsiloxan (PDMS) folie, med mikrometer til millimeter størrelse gennemgående huller forseglet på en cellekultur substrat til fysisk indeholde ECM overtræk og efterfølgende podet hPSCs. Som stencil mønster virker ved fysisk at indskrænke det sted, hvor hPSC kan få adgang til og vedhæfte direkte til den underliggende ECM substrat, denne metode er kompatibel med forskellige substrater, der kan understøtte hPSC kulturer. De eneste requirement er, at valget af stencil materiale kan danne et reversibelt tætning med substratet. Disse substrater indbefatter konventionelle vævskulturpolystyren (TCPS) 17, ligand konjugerede substrater 18, samt elastomere substrater med afstemmelig stivhed (f.eks., PDMS) 19. Denne metode giver også belægning af forskellige ECM, såsom vitronectin (eller VTN protein), laminin og kælder membran matricer (f.eks Matrigel og Geltrax) at give mulighed for en ordentlig vedhæftning og differentiering af hPSCs. Derfor kan vi overfører optimerede ECM-substrat konfigurationer til en specifik hPSC linje til stencil micropatterning for optimal celle-matrix adhæsion, overlevelse og differentiering. For nylig er også rapporteret en lignende metode til direkte hepatisk differentiering ved micropatterning hESCs anvendelse af poly (methylmethacrylat) (PMMA) mikro-stencil grupper 16. Cell stencils kan være fremstillet af forskellige materialer, herunder opfyldtals 20,21, poly (p-xylylen) polymerer 22,23, PMMA 16 og mest almindeligt, PDMS 24-28. Silicium og poly (p-xylylengrupper) polymerer stencils kræver direkte ætsning af de gennemgående huller med specialudstyr 20-23, hvilket begrænser deres tilgængelighed for biologiske brugere. PDMS stencils kan fremstilles ved forskellige metoder afhængigt af funktionen størrelse, der kræves, hvilket typisk fra 3 um til 2.000 um 11,26-29. Hvis der ønskes små features, kan tynde stenciling ark fremstilles ved pressestøbning PDMS pre-polymer på en mikrofabrikeret silicium skabelon indeholdende relieffer af de micropatterns 28. For funktioner&gt; 1.000 um, en CO 2 laserskærer giver en nem og billig metode til direkte skære mønstre på en præ-støbt PDMS ark under stencil opspind. Genanvendeligheden af ​​PDMS stencils gør dem også omkostningseffektivt at gennemføre en række forsøg med tilstrækkelig konsistens. <p class = &quot;jove_content&quot;&gt; Her præsenterer vi den detaljeret metode til fremstilling af en PDMS stencil med 1.000 um funktioner ved laserskæring og generering af hESC micropatterns. Disse hESC micropatterns blev anvendt til at modulere omfanget af integrin og E-cadherin medierede adhæsioner inden for en sammenhængende embryonale koloni for at undersøge, hvordan rumlig polarisering af celleadhæsion resulterede i celleskæbne 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 artikel beskriver vi fremstilling af en celle stencil ved hjælp af en laser cutter til at generere 1.000 um funktioner. Stencilen bestod af 2 dele: en tynd stenciling ark (ca. 100-200 um tyk) indeholdende micropattern gennemgående huller, og en PDMS pakning til at indeholde ECM overtræksopløsningen eller celle suspension. Her blev 127 um og 2 mm tykke kommercielt tilgængelige PDMS plader anvendt som stenciling ark og pakning henholdsvis. Andre fremgangsmåder til fremstillin...

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

Humane pluripotente stamceller (hPSCs) har den iboende evne til at differentiere og selv-organisere sig i forskellige væv mønstre; selvom dette kræver præsentation af rumlige miljømæssige gradienter. Vi præsenterer stencil micropatterning som en enkel og robust metode til at generere biokemiske og mekaniske gradienter til styring hPSC differentieringsmønstre.

Stencil Micropatterning for Human pluripotente stamceller til Sondering Spatial Organisation af differentiering 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

Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering 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

Samlet Size optimering i Microwells for suspension-baserede Cardiac Differentiering af menneskelige pluripotente 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

Hurtig neuronal differentiering af induceret pluripotente stamceller til 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

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente 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 Differentiering af pluripotente stamceller til NKX6-1<sup&gt; +</sup&gt; Pankreatisk progenitorer

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 differentiering af humane pluripotente stamceller til trofoblastceller

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

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente 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 differentiering af humane pluripotente 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 Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

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

Kortlægning af den emergente rumlige organisering af pattedyrsceller ved hjælp af Mikromønstre og kvantitativ billeddannelse

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