Name: epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration
Uploaded: 2016-03-18T14:00:00
Description: Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

E.M.S. was supported by grants from the National Institutes of Health (NIH) [grant number R01HL120919]; the American Heart Association [grant number 10SDG4350046]; University of Rochester CTSA award from the NIH [grant number UL1 TR000042]; and startup funds from the Aab CVRI. M.A.T. was supported in part by a grant to the University of Rochester School of Medicine and Dentistry from the Howard Hughes Medical Institute Med-Into-Grad Initiative; and an Institutional Ruth L. Kirschstein National Research Service Award from the NIH [grant number GM068411].

NOTE: All experimentation with mice were approved by the University Committee on Animal Resources at the University of Rochester.
1. Epicardial Outgrowth Culture Assay (Figure 1A)
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 5 ml medium A for primary epicardial cell isolation by supplementing Medium 199 (M199) with 5% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep).</li>
		<li>Pre-warm medium A and 100 ml Hanks&#39; Balanced Salt Solution (HBSS) in a 37 &#176;C water bath.</li>
		<li>Allow coll...

Here we outline detailed methods to isolate primary epicardial cell by outgrowth and track EPDC migration in ex vivo heart cultures. For outgrowth cultures, the appropriate time to remove the epicardium-depleted heart varies slightly between experiments. Periodic monitoring of the explants after an overnight incubation is recommended to gauge the extent of epicardial outgrowth and to extract the heart before fibroblasts appear. To ensure purity, hearts should be removed within 24 hr of explanting to prevent accu...

The epicardium is a single layer of mesothelial cells that lines the heart and impacts cardiac development, maturation and repair. Through a highly coordinated exchange of paracrine signals, the intimate dialogue between the myocardium and epicardium is indispensable for cardiac growth and the formation of non-myocyte cardiac lineages1. Epicardial-derived cells (EPDC) emerge from a subset of epicardial cells through an epithelial-to-mesenchymal transition (EMT)2, invade the subepicardial space and underlying myocardium, and differentiate largely into coronary vascular mural cells and cardiac fibroblasts, and to a lesser extent, endothelial cells and cardiomyocytes3-9.
The mechanisms regulating epicardial EMT and EPDC invasion have been attributed to the coordinated action of various molecules including secreted ligands10-13, cell surface receptors and adhesion molecules14,15, regulators of apical-basal polarity16,17, small GTPases18,19, and transcription factors2,20,21. While many of the molecular effectors of EPDC migration have been defined, a better understanding of the physiological signals that stimulate EPDC mobilization in the embryo may accelerate the development of strategies to manipulate this process in the adult for improved cardiac repair.
Studies aimed at identifying novel regulators of EPDC mobilization rely on purification of this cell population or tracing the migration of individual epicardial cells. One or a combination of marker genes, including Wt1, Tcf21, Tbx18, and/or Aldh1a2, is commonly used to identify the fetal epicardium1. However, the use of these markers to track migrating EPDC is not optimal as expression of epicardial markers wanes over the course of heart development and is often lost when epicardial cells undergo transdifferentiation into mesenchymal cells.
The application of the Cre/loxP system, in conjunction with lineage-tracing reporters, has been useful in permanently labeling the epicardium and its descendants during heart development and following ischemic injury in the adult7,22,23. Several epicardial-restricted Cre lines have been generated and are widely used to label EPDC and for conditional gene deletion strategies1. These studies have led to the characterization of various epicardial lineages, and the identification of critical mediators of EPDC motility and differentiation. However, accumulating evidence also suggests the epicardium is a heterogeneous population of progenitor cells4,24,25. Thus, only a subset of epicardial cells will be targeted using a given Cre driver.
In order to label the entire epicardium regardless of Cre specificity, a number of laboratories have utilized outgrowth culture systems or ex vivo organ culture methods to faithfully isolate or label epicardial cells without depending upon the expression of a genetic lineage markers26-28. For ex vivo migration studies, embryonic hearts are isolated prior to epicardial EMT and cultured in medium supplemented with a green fluorescent protein (GFP)-expressing adenovirus (Ad/GFP)9,18. This approach allows for efficient labeling of the entire epicardium rather than a subset of cells by Cre-mediated recombination. Heart cultures are subsequently exposed to known inducers of EMT to stimulate the mobilization of EPDC28,29. Ex vivo and in vivo analyses are complemented by in vitro epicardial explant cultures, which are a particularly useful approach for exploring detailed mechanisms driving EPDC migration.
Here, we describe methods for isolating primary epicardial cells for in vitro studies of epicardial EMT, as well as an organ culture system for ex vivo analyses of EPDC motility. We have recently demonstrated the utility and robustness of this method by genetically modulating the myocardin-related transcription factor (MRTF)/serum response factor (SRF) signaling axis to manipulate actin-based migration of EPDC9. While our findings highlight one signaling pathway necessary for regulating EPDC migration, these methods are suitable for unraveling the mechanisms that collectively orchestrate EPDC migration and differentiation. Furthermore, the explant and ex vivo culture systems could be implemented in functional screens to identify novel regulators of EPDC mobilization for therapeutic applications in cardiac regeneration.

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			<td height="40" style="height:40px;width:244px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:163px;">Hyclone&#160;</td>
			<td style="width:141px;">SH3002201</td>
			<td style="width:203px;">DMEM</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SH3003102</td>
			<td style="width:203px;">HBSS</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Medium 199</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SH3025301</td>
			<td style="width:203px;">M199</td>
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			<td height="40" style="height:40px;width:244px;">Dulbecco&#8217;s Phosphate-Buffered Saline&#160;</td>
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			<td height="20" style="height:20px;width:244px;">Multiwell 24-well plates</td>
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			<td style="width:141px;">170-8880</td>
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			<td height="20" style="height:20px;width:244px;">Protease inhibtor cocktail tablets</td>
			<td style="width:163px;">Roche</td>
			<td style="width:141px;">11836170001</td>
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			<td height="20" style="height:20px;width:244px;">Alexa Goat-anti-rabbit 488</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A11008</td>
			<td style="width:203px;">Secondary antibody</td>
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			<td height="20" style="height:20px;width:244px;">Alexa Goat anti-rabbit 594</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A-11012</td>
			<td style="width:203px;">Secondary antibody</td>
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			<td style="width:141px;">A-11005</td>
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			<td style="width:203px;">monoclonal antibody, clone 1A4</td>
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			<td height="40" style="height:40px;width:244px;">Mouse anti-Wilms tumor 1 (WT1)</td>
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			<td>MA1-46028</td>
			<td style="width:203px;">monoclonal antibody, clone 6F-H2</td>
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			<td height="20" style="height:20px;width:244px;">Rabbit anti-ZO1</td>
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			<td>40-2200</td>
			<td style="width:203px;">polyclonal antibody</td>
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			<td height="20" style="height:20px;width:244px;">Rabbit anti-Collagen Type 4</td>
			<td style="width:163px;">Millipore</td>
			<td style="width:141px;">AB756P</td>
			<td style="width:203px;">polyclonal antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:244px;">4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride&#160;</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">D1306</td>
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			<td height="20" style="height:20px;width:244px;">Fluorescent mounting medium&#160;</td>
			<td style="width:163px;">DAKO</td>
			<td style="width:141px;">S3023</td>
			<td style="width:203px;"></td>
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			<td height="60" style="height:60px;width:244px;">16% Paraformaldehyde solution</td>
			<td style="width:163px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">15710</td>
			<td style="width:203px;">PFA diluted to 4% in DPBS. Caution, hazardous material</td>
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			<td height="20" style="height:20px;width:244px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:141px;">S0389</td>
			<td style="width:203px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Tissue Freezing Medium</td>
			<td style="width:163px;">Triangle Biomedical Sciences</td>
			<td style="width:141px;">TFM-B</td>
			<td style="width:203px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Pap pen</td>
			<td style="width:163px;">DAKO</td>
			<td style="width:141px;">S2002</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Disposable mictrotome blades&#160;</td>
			<td style="width:163px;">Sakura Finetek</td>
			<td style="width:141px;">4689</td>
			<td style="width:203px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Microscope slides</td>
			<td style="width:163px;">Globe Scientific</td>
			<td style="width:141px;">1358W</td>
			<td style="width:203px;">positively charged, 25 x 75 x 1mm</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cryostat&#160;</td>
			<td style="width:163px;">Leica</td>
			<td style="width:141px;">CM1950</td>
			<td style="width:203px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Forceps</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:141px;">11295-00</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Surgical Scissors</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:141px;">91460-11</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Disposable base molds</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:141px;">22-363-553</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ketamine</td>
			<td style="width:163px;">Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Xylazine</td>
			<td style="width:163px;">Akorn</td>
			<td>NADA 139-236</td>
			<td style="width:203px;"></td>
		</tr>
	</tbody>
</table>

epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

The epicardium can be efficiently isolated using an outgrowth culture assay by taking advantage of its exterior location and exploiting the developmental plasticity and intrinsic migratory behavior of EPDC. Murine embryonic hearts are isolated at E11.5 prior to epicardial EMT and cultured dorsal side down on collagen-coated chamber slides26 (Figure 1A, B). Explanted hearts will continue to contract; however, the epicardium will adhere to the collagen matrix and...

Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

The overall goals of the outgrowth culture method and the ex vivo migration assay are to isolate primary cultures of epicardial cells for studying epithelial to mesenchymal transitions and to assess the functional migration of epicardial derived cells, respectively. The main advantage of these techniques is that they facilitate the isolation of primary cultures from the epicardium and the testing of the intrinsic behaviors of epicardial derived progenitor cells. The implications of this technique extend toward the development of strategies for manipulating epicardial derived cell biology in cardiac regenerative medicine.In preparation of epicardial cell isolations, pre-warm collagen coated chamber slides to room temperature and add 100 microliters of medium A per well. Remove media after coating chambers ensuring no pooled media remains. To isolate primary epicardial cells first harvest mouse embryos at 11.5 days post coitum.Use forceps to pinch the thoracic wall of embryos and tear the tissue apart at the midline, exposing the chest cavity. Next place the forceps behind the the heart and grasp the outflow tract. Pull the heart away from the embryo and place the cardiac tissue, dorsal side down, in a single well of a collagen coated slide.When all of the hearts have been placed in individual wells, incubate the chamber slide for 30 minutes at 37 degrees Celsius and 5%CO2 to allow the tissue to adhere to the collagen matrix. At the end of the incubation, carefully add 20 to 50 microliters of pre-warmed explant medium A around each heart and return the slide to the cell culture incubator for approximately 24 hours to facilitate the epicardial outgrowth. It's important to add the appropriate amount of medium to the explants, as too much may result in tissue detachment and too little medium may result in dehydration, diminishing the quality of the outgrowths.Outgrowths can be observed as early as three to four hours of culture and by 24 hours, consist primarily of mesothelial cells that display a cobblestoned morphology with a minimal mesenchymal cell contamination. Care should be taken to remove hearts before invasion of mesenchymal cells contaminate the epicardial cell outgrowth. The next day, use forceps to carefully transfer the epicardium-depleted hearts to a one point five milliliter tube.Then add 800 microliters of Trizol and store the samples at negative 80 degrees Celcius for later downstream analysis. Then wash each well with DPBS to remove the blood cells and excess cellular debris. Incubate the outgrowth cultures with 500 microliters of explant medium B supplemented with adenovirus expressing Cre recombinase for 24 hours in a cell culture incubator.For epicardial labeling to track subsequent cell migration isolate the embryonic hearts from day 12.5 post coitum pregnant dams, as just demonstrated, and place them into individual wells of a 24-well plate containing pre-warmed ex vivo culture medium. Incubate the hearts at 37 degrees Celcius and 5%CO2 with gentle rocking. While the hearts are incubating, resuspend three point oh times 10 to the sixth pfu per milliliter of adenovirus GFP and 12 milliliters of 37 degrees Celcius ex vivo culture medium.Next transfer six milliliters of the virus-supplimented culture medium into a new 15 milliliter tube and add adenovirus Cre to a final concentration of one point five times 10 to the sixth pfu per milliliter. Using a P1000 pipette carefully remove the culture medium from each heart and add the adenovirus GFP adenovirus Cre solution to each well. Then return the plate to the cell culture incubator for 24 hours with gentle rocking.For the epithelial to mesenchymal induction the next day use a P1000 pipette to remove the medium from each well. Carefully rinse the hearts with one milliliter of DPBS. Then discard the wash and replenish the hearts with freshly prepared ex vivo culture medium, containing TGF beta one and platelet derived growth factor BB, and incubate for 24 hours in the cell culture incubator with gentle rocking.Epicardial cell explants display a robust expression of epicardial and mesothelial markers and a low expression of cardiomyocyte genes compared to the epicardium-depleted hearts. To further verify their identity, epicardial cell cultures are fixed 48 hours after the removal of the heart and co-stained with antibodies against Wilms'tumor one and ZO-1 to label the mesothelial cells and intercellular tight junctions, respectively. After 24 hours of stimulation the epicardial cells adopt a mesenchymal phenotype with a reduced ZO-1 staining and a robust de novo formation of smooth muscle alpha-actin positive stress fibers, particularly at the periphery of the culture.In contrast, confluent epicardial cells with more defined inter-cellular contacts at the center of a monolayer display smooth muscle alpha-actin associated with cortical regions. Deletion of the miocardin related transcription factor signaling axis genes by Cre-mediated excision of fluxed alleles attenuates the migration of epicardial derived cells into the sub-epicardial space. Conversely, the forced expression of myocardin related transcription factor A results in an increased migration and disruption of the collagen IV basement membrane by the epicardial derived cells.Once mastered, each technique can be completed in 48 to 72 hours if performed properly. While attempting this procedure it's important to isolate embryos at the appropriate developmental time stages to optimize epicardial cell outgrowth from tissue explants and to label the epicardium prior to epicardial derived cell migration. Following this procedure other methods, like genetic manipulation or chemical modifications can be performed to answer questions about identifying novel regulators of epicardial derived cell mobilization and differentiation.

epicardial-outgrowth-culture-assay-ex-vivo-assessment-epicardial

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

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

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

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

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

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...