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

This work is supported by NUS Start up grant (R-397-000-192-133) and ETPL Gap Fund (R-397-000-198-592). G.S. is a NUS Research scholar. Authors would like to thank Dr. Jiangwa Xing for her technical support on cell micropatterning.

NOTE: This protocol describes the fabrication of PDMS stencil with 1,000 &#181;m patterns by laser-cutting and micropatterning of the hESC line, H9 using the PDMS stencil.
1. Design and Fabrication of PDMS Stencil for Micropatterning
<ol>
	<li>Design the stenciling sheet with through-holes of the desired geometry and size (e.g., 1,000 &#181;m circles) and the stencil gasket using computer-aided design software10.</li>
	<li>Laser-cut the stenciling sheet and gasket on 120-150 &#181;m and 2 mm thick polydimethylsiloxane (PDMS) sheets respectively using a CO2 laser-cutter10.</li>
	<li>Bond the PDMS gasket with the PDMS stencil sheet using liquid uncured PDMS and bake at 60 &#176;C for 3-4 hr to obtain the PDMS stencil for micropatterning.</li>
	<li>Sterilize the PDMS stencil by autoclaving at 120 &#176;C for 30 min and drying in an oven before use.</li>
</ol>
2. hESCs Maintenance
<ol>
	<li>Culture the hESC lines in a feeder-free maintenance medium on 1&#215; hESC-qualified basement membrane matrix coated cell culture plates at 37 &#176;C and 5% CO2.</li>
	<li>Passage the hESCs when they are approximately 70% confluent by 3 min incubation at 37 &#176;C with dispase treatment and mechanically dissociating the hESC colonies into 100-200 &#181;m clumps. Plate the hESCs clumps at a density of 30-50 clumps per 9.6 cm2 of growth area on basement membrane matrix-coated tissue culture polystyrene at 1:6 splitting ratio.</li>
</ol>
3. Stencil Micropatterning of Human Embryonic Stem Cells
<ol>
	<li>Preparation prior to cell patterning
	<ol>
		<li>Seal a PDMS stencil onto a 60 mm Petri dish by dispensing 700 &#181;l 70% analytical grade ethanol in ultrapure water into the Petri dish and placing the stencil on top.</li>
		<li>Place the Petri dish inside biosafety cabinet overnight to let the ethanol dry.</li>
		<li>Check no bubble exists underneath the stencil to ensure that the stencil forms a good seal with the Petri dish. This prevents leakages of ECM coating solution. Seal the Petri dish and store under sterile conditions until it is ready for cell seeding.</li>
		<li>Prepare aliquots of hESC-qualified basement membrane matrix according to the Certificate of Analysis. Ensure that each aliquot yields 1&#215; hESC-qualified basement membrane matrix solution when diluted in 25 ml of Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) according to manufacturer&#39;s product sheet.</li>
		<li>Prepare the ECM coating solution by adding one aliquot of hESC-qualified basement membrane matrix into 16.7 ml of DMEM/F12 to make 1.5&#215; hESC-qualified basement membrane matrix solution. Keep all the ECM coating solution on ice to prevent gelation.</li>
		<li>Supplement hESC maintenance medium with 10 &#181;M ROCK inhibitor (Y27632).</li>
	</ol>
	</li>
	<li>ECM coating on the stenciled substrate
	<ol>
		<li>Treat the Petri dish with 100 W O2 plasma for 90 sec. To ensure sterility, only open the Petri dish cover inside the plasma chamber prior to plasma treatment, and quickly cover back the Petri dish after completion of treatment. This facilitates surface wetting and prevents air bubble formation in the micropattern through-holes during the addition of ECM coating solution.</li>
		<li>Add 450 &#181;l of 1.5&#215; hESC-qualified basement membrane matrix solution to cover the whole stencil. Seal the Petri dish with self-sealable film, such as Parafilm, to prevent the solution from drying out, and incubate for 5 hr at 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Seeding hESCs onto the stenciled substrate
	<ol>
		<li>Examine hESC colonies in 6-well plate to identify the differentiated cell areas showing loss of typical hESC morphology (e.g., loss of rounded, tightly packed epithelial morphology, high nucleus/cytoplasm ratio with prominent nucleoli). Remove differentiated regions using a vacuum aspirator.</li>
		<li>Wash twice with 2 ml DMEM/F12 per well.</li>
		<li>Add 1 ml of digestive enzymes, such as Accutase, per well of 6-well plate and incubate at 37 &#176;C for 8 min. Tap the plate gently to detach all colonies from substrate.</li>
		<li>Rinse each well with at least 4 ml of DMEM/F12 per 1 ml of digestive enzymes and collect the cell suspension into 15 ml conical tube.</li>
		<li>Centrifuge the cell suspension at 200 &#215; g for 3 min at room temperature.</li>
		<li>Aspirate to remove the supernatant and add 400 &#181;l of hESC maintenance medium supplemented with ROCK inhibitor (ROCKi) to re-suspend the cells. Pipette the cell suspension up and down 3 times gently to break clumps into single cells.</li>
		<li>Mix the single cell suspension well and dilute 10 &#181;l of cell samples into 190 &#181;l of DMEM/F12 (1:20 dilution). Use a hemocytometer to determine the cell density in the stock cell suspension.</li>
		<li>Calculate the required cell seeding density for a given stencil. 
		NOTE: For example, we have experimentally determined the cell seeding density to obtain a confluent monolayer of single cells is approximately 4,444 cells/mm2. Thus, a stencil with an area of 450 mm2 and seeding volume of 400 &#181;l will require a cell suspension to be at a density of 2 million cells / 400 &#181;l.</li>
		<li>Dilute the stock cell suspension to the required seeding density (see step 3.3.8 NOTE) with hESC culture medium supplemented with ROCKi.</li>
		<li>Add a designated volume of cell suspension containing the required number of cells into each stencil and leave the Petri dish undisturbed in hood for 5 min at room temperature to allow cells to settle.</li>
		<li>Transfer the Petri dish into incubator and incubate for 1 hr to allow for cell attachment. Take care to keep the Petri dish level during the transfer process so that cells remain as a monolayer in the stencil.</li>
	</ol>
	</li>
	<li>Stencil removal and passivation of unpatterned substrate
	<ol>
		<li>Examine the Petri dish under a microscope to check if cells are properly attached onto the underlying substrate.</li>
		<li>Aspirate away cell suspension from the stencil.</li>
		<li>Add 2 ml/dish of 0.5% cell culture compatible non-ionic surfactant in DMEM/F12 to the area surrounding the stencil.</li>
		<li>Use a pair of autoclaved forceps to gently peel off the stencil. Swirl the non-ionic surfactant solution around as the stencil is peeled off to prevent the cells from drying out. Visually observe the micropatterned-cells in the Petri dish.</li>
		<li>Incubate the micropatterned-cells in 0.5% non-ionic surfactant-DMEM/F12 solution for 10 min at 37 &#176;C.</li>
		<li>Aspirate away the non-ionic surfactant-DMEM/F12 solution. Wash 3 times with 2 ml/dish of DMEM/F12.</li>
		<li>Add 2 ml/dish of hESC maintenance medium supplemented with ROCKi and incubate at 37 &#176;C overnight.</li>
		<li>After overnight incubation, aspirate hESC maintenance medium supplemented with ROCKi, wash once with DMEM/F12 and induce mesoendoderm differentiation by adding 2 ml/dish of differentiation medium supplemented with 100 ng/ml Activin, 25 ng/ml BMP4 and 10 ng/ml FGF2.</li>
		<li>Evaluate the micropatterns using phase contrast imaging and compute the average Brachyury (T) intensity profiles for each pattern as described previously8.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

Fabrication of micropatterning stencils
Stencil micropatterning provides an ideal method to generate hPSC micropatterns for investigating niche-mediated differentiation patterning. The key advantage of stencil patterning over other micropatterning techniques, such as microcontact printing and photopatterning, is that it does not require surface modification and can be implemented on conventional TCPS substrates. Therefore, optimized culture media and ECM coatings for ...

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are widely exploited in regenerative medicine as well as experimental modeling of normal and diseased organogenesis because of their differentiation potential into cell lineages of all three germ layers1,2. The differentiation fates of hPSCs are highly sensitive to local environmental factors that can modulate autocrine or paracrine signaling1 as well as mechanotransduction processes mediated by physical cues3-5. Cell micropatterning encompasses a set of techniques that have been developed to spatially organize the geometry and location of a cell population as a mean to control the local cellular microenvironment, such as cell-cell interactions6 and cell-matrix interactions3. In the context of hPSCs, cell micropatterning has been employed to gain significant insights into how niche-dependent autocrine signaling modulates hESC pluripotency-differentiation decisions7 and organization into early embryonic differentiation patterns6. 2D and 3D micropatterned hPSCs have been used to control the colony size of multicellular patterns, which in turn influenced differentiation decisions into the three germ layers8,9. We have employed multicellular hPSC micropatterns to modulate the extent of cell-cell and cell-matrix interactions within a hPSC colony to probe how integrin-E-cadherin crosstalk can give rise to cell fate heterogeneity10. The demonstrations from the above reports open new avenues towards the application of multicellular micropatterns of hPSCs as experimental models for drug toxicity screening for developmental diseases11, to study the effect of growth factors and hormones during tissue or organ development, and to unravel the formation of tissue patterns.
A myriad of cell micropatterning techniques have been developed as reviewed by Falconnet et. al.12 but only a handful, such as micro-contact printing7,8,13, microwell culture14,15, photopatterning6 and microstencils16 have been successfully implemented with hPSCs. The challenge with micropatterning hPSCs lies in their vulnerability and a stringent requirement of specific extracellular matrices (ECM) and growth conditions for cell attachment and survival. For 2D hPSC patterns, micro-contact printing is one of the most common methods to generate hPSC micropatterns on tissue culture and glass substrates13. The method can be used to pattern common ECM used in hPSC culture, including laminin and basement membrane matrices, such as Matrigel. However, it typically requires a two-step coating process aided by Poly-D-Lysine, and needs specific inert atmospheric and humidity conditions to make stable ECM micropatterns for hPSCs to attach on6,13. The foremost consideration of each micropatterning method is whether the surface modification regime can generate hPSC-adhesive ECM patterns at the desired geometrical resolution while minimizing unspecific cell attachment to the surrounding areas.
Here, we report the use of stencil micropatterning as a simple method to generate hPSC micropatterns without additional surface modification steps prior to the generation of adhesive ECM patterns for hPSCs to attach on. The cell stencil comprises of a thin membrane, e.g., polydimethylsiloxane (PDMS) sheet, with micron to millimeter size through-holes sealed onto a cell culture substrate to physically contain ECM coatings and subsequently seeded hPSCs. As stencil patterning works by physically restraining the location where hPSC can access and attach directly to the underlying ECM coated substrate, this method is compatible with various substrates that can support hPSC cultures. The only requirement is that the choice of stencil material can form a reversible seal with the substrate. These substrates include conventional tissue culture polystyrene (TCPS)17, ligand conjugated substrates18, as well as elastomeric substrates with tunable stiffness (e.g., PDMS)19. This method also allows coating of different ECM, such as vitronectin (or VTN protein), laminin and basement membrane matrices (e.g., Matrigel and Geltrax) to allow for proper attachment and differentiation of hPSCs. Therefore, we can transfer optimized ECM-substrate configurations for a specific hPSC line to stencil micropatterning for optimal cell-matrix adhesion, survival and differentiation. Recently, a similar method has also been reported to direct hepatic differentiation by micropatterning hESCs using poly(methyl methacrylate) (PMMA) micro-stencil arrays16.
Cell stencils can be fabricated from different materials, including metals20,21, poly(p-xylylene) polymers22,23, PMMA16 and most commonly, PDMS24-28. Silicon and poly(p-xylylene) polymers stencils require direct etching of the through-holes with specialized equipment20-23, which limits their accessibility to biological users. PDMS stencils can be fabricated by different methods depending on the feature size required, which typically ranges from 3 &#181;m to 2,000 &#181;m11,26-29. If small features are desired, thin stenciling sheets can be produced by press molding PDMS pre-polymer on a microfabricated silicon template containing reliefs of the micropatterns28. For features &#62; 1,000 &#181;m, a CO2 laser cutter provides an easy and low cost method to directly cut the patterns on a pre-casted PDMS sheet during stencil fabrication. The recyclability of PDMS stencils also makes them cost-effective to conduct a series of experiments with sufficient consistency.
Here, we present the detailed methodology for the fabrication of a PDMS stencil with 1,000 &#181;m features by laser cutting and the generation of hESC micropatterns. These hESC micropatterns were used to modulate the extent of integrin and E-cadherin mediated adhesions within a cohesive hESC colony so as to investigate how spatial polarization of cell adhesion resulted in cell fate heterogeneity10.

<table><tbody><tr><td>&amp;nbsp;2 mm thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.005</td><td>Used to form reservoir for stencil</td></tr><tr><td>120-150 &amp;mu;m thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.040</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>60 mm Petri dish</td><td>Nunc Nunclon Delta</td><td>150326</td><td>Substrate for micropatterning</td></tr><tr><td>Accutase</td><td>Accutase, Merck Millipore, Singapore</td><td>SCR005</td><td>Enzyme to break H9 cells into single cells</td></tr><tr><td>Activin&amp;nbsp;&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-AC-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>BMP4&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-BP-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>Plasma system&amp;nbsp;</td><td>Femto Science, Korea</td><td>CUTE-MP</td><td>For plasma oxidation of stencil</td></tr><tr><td>Dispase</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>7923</td><td>Enzyme used to weaken the cell-ECM adhesion during passaging</td></tr><tr><td>DMEM/F12</td><td>GIBCO, USA</td><td>11330032</td><td>Basal medium for H9 cells</td></tr><tr><td>FGF2</td><td>R&amp;amp;D Systems, Singapore</td><td>233&amp;ndash;FB&amp;ndash;025</td><td>Growth factor for H9 differentiation</td></tr><tr><td>H9 cell line</td><td>WiCell Research Institute, Inc., USA</td><td>WA09</td><td>Human embryonic stem cells</td></tr><tr><td>hESC-qualified basement membrane matrix</td><td>Matrigel, BD Biosciences, Singapore</td><td>354277</td><td>Extra-cellular matrix coating to support growth of H9 cells</td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems, Singapore</td><td>DMi1</td><td>For capturing bright-field images</td></tr><tr><td>Laser cutter</td><td>Epilog Helix 24 Laser System</td><td></td><td>Used to generate through holes in PDMS sheet</td></tr><tr><td>mTeSR&lt;sup&gt;&amp;trade;&lt;/sup&gt;1 medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5850</td><td>Maintainence medium for H9 cells</td></tr><tr><td>PDMS&amp;nbsp;</td><td>SYLGARD&amp;reg; 184, Dow Corning Co., USA</td><td>3097358-1004</td><td>Used for sticking the PDMS stencil and reservior</td></tr><tr><td>ROCKi Y27632</td><td>Calbiochem, Merck Millipore, Singapore</td><td>688000</td><td>Maintains H9 cells as single cells&amp;nbsp;</td></tr><tr><td>STEMdiff&lt;sup&gt;&amp;trade;&lt;/sup&gt; APEL&lt;sup&gt;&amp;trade;&lt;/sup&gt; medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5210</td><td>Differentiation medium for H9 cells</td></tr><tr><td>Polyethylene terephthalate film</td><td>SureMark Singapore</td><td>SQ-6633</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>Cell culture compatible non-ionic surfactant</td><td>Pluronic acid F-127, Sigma, Singapore</td><td>P2443</td><td>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 this paper, we describe the fabrication of a cell stencil by using a laser cutter to generate 1,000 &#181;m features. The stencil was composed of 2 parts: a thin stenciling sheet (approximately 100-200 &#181;m thick) containing the micropattern through-holes, and a PDMS gasket to contain the ECM coating solution or cell suspension. Here, 127 &#181;m and 2 mm thick commercially available PDMS sheets were used as the stenciling sheet and gasket respectively. Other methods for preparing P...

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

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

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

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Research

JoVE Journal

Developmental Biology

8.4K Views.  National University of Singapore.  Menselijke pluripotente stamcellen (hPSCs) hebben de intrinsieke vermogen om onderscheid te maken en zichzelf organiseren in verschillende weefsel patronen; hoewel dit vereist de presentatie van ruimtelijke milieu-hellingen. Wij presenteren stencil micropatterning als een eenvoudige en robuuste methode om biochemische en mechanische gradiënten genereren voor het besturen van HPSC differentiatie patronen. 

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

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

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

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

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

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

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

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

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and 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 when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

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

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Biology

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Bioengineering

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Monitoring Pluripotent Stem Cell Differentiation by Image-Based ML Strategy

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

Author Spotlight: Enhancing PSC-to-Functional Cell Differentiation Using ML Models Based on Live-Cell Bright-Field Imaging

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...

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

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