Name: a pipeline to characterize structural heart defects in the fetal mouse
Uploaded: 2022-12-16T13:45:00
Description: Watch this Scientific Journal Video about A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse at JoVE.com

The use of mice for these studies is necessary as mice have four-chambered hearts that can mimic human CHDs. Mice were provided veterinary care and housed in the institution&#39;s Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal care facility. Strict protocols were followed to minimize the mice&#39;s discomfort, stress, pain, and injury. Mice were euthanized using CO2 gas, which is acceptable for small rodents according to the American Veterinary Medical Associa...

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Genetically modified mice have been used to understand the pathomechanisms of congenital heart defects. The protocols we provide in this study attempt to streamline and standardize the process of assessing murine fetal heart defects. However, there are critical steps to note during the protocol. Mouse embryos grow significantly during each day of gestation, and the correct time to harvest a mouse can be determined by performing a fetal echocardiogram accurately. The fetal echocardiogram can be used to screen the fetal ca...

Congenital heart diseases (CHDs) are the most common neonatal birth defect1,2, affecting about 0.8%-1.7% of neonates and resulting in significant neonatal mortality and morbidity3. A genetic etiology is strongly indicated with CHDs4,5. Genetically modified mouse models have been used widely to understand the complexity of CHDs and the mechanisms that cause them due to the mice having four-chamber hearts and comparable cardiac developmental DNA sequences in mouse and human fetuses6. Identifying the phenotype of the mouse mutants is the fundamental first step in characterizing the function of the targeted gene. Mouse models expressing gene dosage effects, in which a single genetic mutation can result in a spectrum of cardiac defects that mimic human CHDs, are important for understanding the complexity of CHDs and the mechanisms that cause them.
This article outlines a pipeline to characterize cardiac phenotypes in mouse models. The applied methods utilize fetal echocardiogram7, followed by necropsy and ECM histopathology7,8, which can display the detailed anatomy of developing murine cardiac phenotypes. A fetal echocardiogram is a noninvasive modality that allows direct visualization of multiple embryos with reasonable imaging resolution. In addition, a fetal echocardiogram provides a quick determination of the total number of embryos in a litter, their developing stages, and the relative orientation and location in the uterine horn. Using a spectral Doppler/color flow, abnormal embryos can be identified based on the structure, the hemodynamic disturbance, the growth restriction, or the development of&#160;hydrops. Since a fetal echocardiogram study is a noninvasive technique, it can be used to scan on multiple days and to observe the changes in hemodynamics or cardiac morphology. Obtaining high-quality imaging of fetal echocardiograms requires practice and skill, as specific heart defects may be missed due to a lack of experience and knowledge. Because of this, a more definitive analysis of cardiac morphology may be obtained through a combination of necropsy and ECM histopathology. Necropsy provides direct visualization of the arch structure, the relative relationships of the aorta and pulmonary artery, the size of the ventricles and atria, the position of the heart relative to the chest, and the bronchopulmonary structures. However, interior features such as the heart valves and wall thickness may be difficult to assess through necropsy alone. Thus, ECM histopathology is recommended for a conclusive diagnosis. ECM histopathology is a high-resolution visualization technique that allows for both 2D and 3D reconstruction of the image stack9. These images are obtained through serial Episcopic fluorescent imaging of a paraffin-embedded sample as it is thinly sectioned at a consistent interval by an automatic microtome. Unlike classical histology, images are captured as a section before it is cut from the block such that all images are captured within the same reference frame. Because of this, the 2D image stack produced by ECM histopathology may easily and reliably be reconstructed in three dimensions. This is done using a DICOM viewer, which allows 3D visualization of the images in the three anatomical planes: coronal, sagittal, and transverse. From these high-resolution 3D reconstructions, a definitive cardiac diagnosis may be made. The application of these three different visualization modalities, either individually or in combination, can provide accurate characterizations of structural heart defects in mouse embryos.

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			<td>Sigma Aldrich</td>
			<td>P3813</td>
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			<td>1.5 mL Eppendorf tubes (or preferred vial for tissue storage)</td>
			<td>SealRite</td>
			<td>1615-5599</td>
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			<td>10% buffered formalin phosphate solution</td>
			<td>Fisher Chemical</td>
			<td>SF100-4</td>
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			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
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			<td>16% paraformaldehyde (PFA) fixative&#160;</td>
			<td>ThermoScientific</td>
			<td>28908</td>
			<td>4% working concentration freshly prepared in 1x PBS at 4 &#176;C</td>
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			<td>50 mL tubes</td>
			<td>Falcon</td>
			<td>352070</td>
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			<td>6&#8211;12 Well plate or 20 mL vial&#160; for embryo storage</td>
			<td>Falcon</td>
			<td>353046</td>
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			<td>Dissecting microscope&#160;</td>
			<td>Leica</td>
			<td>MDG36</td>
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			<td>Dissecting Pins (A1 or A2 grade)</td>
			<td>F.S.T</td>
			<td>26002-15</td>
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			<td>Dissecting Plate&#160;</td>
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			<td>FB0875713</td>
			<td>Petri dish with paraffin base</td>
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			<td>Embedding molds</td>
			<td>Sakura</td>
			<td>4133</td>
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			<td>Extra narrow scissors (10.5 cm)</td>
			<td>F.S.T</td>
			<td>14088-10</td>
			<td>1&#8211;2 pairs&#160;</td>
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			<td>Fiji application/Image J</td>
			<td>NIH</td>
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			<td>Fiji.sc</td>
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			<td>Fine tip (0.05 mm x 0.01 mm) Dissecting Forceps (11 cm)</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td>2 Pairs</td>
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			<td>Hot forceps&#160;</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td>For orientation of embryos</td>
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			<td>Industrial Marker for Wax Blocks&#160;</td>
			<td>Sharpie</td>
			<td>2003898</td>
			<td>Formatted for labratory use</td>
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			<td>Jenoptik ProgRes C14plus Microscope Camera&#160;</td>
			<td>Jenoptik</td>
			<td>017953-650-26</td>
			<td></td>
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			<td>Jenoptik ProgRess CapturePro acquisition software</td>
			<td>Jenoptik</td>
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			<td>jenoptik.com</td>
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			<td>Large glass beaker&#160;</td>
			<td>Fisher Scientific</td>
			<td>S111053</td>
			<td>For melting paraffin</td>
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			<td>Leica M165 FC binocular microscope (16.5:1 zoom optics)</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
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			<td>OsiriX MD Version 12.0</td>
			<td>OsiriX</td>
			<td></td>
			<td>osirix-viewer.com&#160;</td>
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			<td>Paraplast embedding paraffin wax</td>
			<td>Millipore Sigma</td>
			<td>1003230215</td>
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			<td>Small glass beaker</td>
			<td>Fisher Scientific</td>
			<td>S111045</td>
			<td></td>
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			<td>Small, perforated spoon (14.5 cm)</td>
			<td>F.S.T</td>
			<td>10370-17</td>
			<td></td>
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			<td>Straight Vannas Scissors (4&#8211;8 mm)</td>
			<td>F.S.T</td>
			<td>15018-10</td>
			<td>A pair</td>
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			<td>Vevo2100 ultrahigh-frequency ultrasound biomicroscope</td>
			<td>FUJIFILM VisualSonics Inc.</td>
			<td>Vevo2100</td>
			<td></td>
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			<td>Xylene</td>
			<td>Fisher Chemical</td>
			<td>UN1307</td>
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a pipeline to characterize structural heart defects in the fetal mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

The mouse embryos with significant hemodynamic defects were noted to be embryonic lethal. A wide variety of CHDs can be identified through the high output, noninvasive fetal echocardiogram using different views (Figure 1).
Septal defects: The most common CHDs are septal defects such as a ventricular septal defect (VSD), an atrioventricular septal defect (AVSD), and an atrial septal defect (ASD)1. VSD or AVSD can be easily v...

Watch this Scientific Journal Video about A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse at JoVE.com

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

This protocol outlines cutting edge methods for generating high resolution imaging data for diagnosing congenital heart defects. We conduct functional assessments with non-invasive fetal echocardiography combined with necropsy followed by episcopal confocal microscopy histopathology to generate serial 2D image stacks and 3D reconstructions for comprehensive CHD diagnosis. This method can elucidate the detailed anatomical changes not only in congenital heart diseases, but also can be used to investigate structural birth defects in other organs including cranial facial defects, limb and skeletal anomalies, as well as defects of the brain and other visceral organs.Demonstrating these procedures will be Meghan Holbrook, Carla Guzman, Peizhao Zhang, and Benjamin Glennon, all researchers from our laboratory. After preparing the mouse for the procedure, warm up the ultrasound gel to a body temperature and apply it generously over the imaging area. Then place the transducer on the abdomen oriented in a horizontal plane and identify the bladder on the screen.Once the bladder is identified, scan cranially from the bladder, look for the fetus, and determine the gestational age by measuring the crown to rump length. Next, use the color doppler to analyze blood flow from the heart. To allow fixative penetration into the internal organs, make incisions in the lateral thorax and abdomen using forceps or dissecting scissors and allow the sample to fix for at least 24 hours in a cold room.Then set up the software to save the images with a name, including the sample's identification, the microscope magnification, and the content of the picture. Pin the sample through its wrists and ankles. Before cutting the skin along the median axis towards the tail, lift the skin in the middle of the neck using forceps and cut the skin toward both the armpits with the scissors.Then cut the skin from the umbilicus to the legs. After embedding the samples, adjust the laser projection position to the center of the specimen on the screen and adjust the zoom knob to 20x. To optimize the resolution, set the gain to 1, 250V.Maximize the blue area using the focus knob, and then reset the gain to approximately 750V for imaging. Next, switch the cutting method to auto. Set the thickness to around 50 micrometers and run the slides.Stop cutting when the lungs and airway are in view. Choose a cutting thickness between eight to 10 micrometers and stop the live view open. Open Microtome Communicator to start imaging.Ensure the temporary storage folder is empty before collecting images. Stop the image collection when no hard structure is visible. Output the temporary stored files into one TIFF image series.For 3D image reconstruction, drag and drop the image files into the image processing software. Once the pop-up window appears, select the links or the files to copy into the database. Once the file is opened, click on the 2D 3D reconstruction tools menu from the toolbar and select 3D MPR.For pixel X resolution, and pixel Y resolution, input the image resolution by giving the zoom used during ECM imaging. For the slice interval, input the slice thickness used to cut during ECM imaging. Next, use the WWWL tool to adjust the window width and window level.Click and drag the tool on the image upward to decrease image brightness and downward to increase it. Click and drag the tool on the image rightward to decrease image contrast and left word to increase it. Drag the images into the desired positions using the pan tool.Use the zoom tool to enlarge or shrink the image and the rotate tool to rotate the image as desired. Now, click and drag the colored axis of the first panel. Notice how rotating this axis changes the orientation of the other two panels.Rotate the three panels axes until the three panels represent coronal, sagittal, and transverse views of the sample. Once all three panels are appropriately positioned, oriented, and brightened, click on the panel representing the coronal view. Then click on Movie Export on the right side of the menu bar.Click on batch and drag the from and to sliders to encompass the entire region of interest. For interval, select the same as thickness option and save the video indicating the views orientation. The echo shows an intact ventricular septum without an intraventricular shunt and normal related great arteries in the normal control, which was confirmed by ECM.The frontal view demonstrates communication between LA, RA, LV, and RV in both echo and ECM, suggesting atrioventricular septal defect. The color flow demonstrates the flow across LV and RV indicating ventricular septal defect. Echo and ECM demonstrated a double outlet of right ventricle with a ventricular septal defect between LV and RV with side by side great arteries.Ultrasound diagnosis of persistent truncus arteriosus demonstrates communication between LV and RV with a single outflow tract overriding both ventricles, also confirmed by ECM at the E14.5 stage. The sections collected from ECM histology can be further processed for immunostaining, hematoxylin and eosin staining and even nucleic acid extraction for genotyping and transcriptional profiling or protein extraction for proteomic analysis. The images from three-dimensional reconstruction can be further processed for assessments of quantitative perimeters such as area or volume.Accurate phenotyping to identify defects in the cardiovascular anatomy is essential for establishing genetically engineered and mutant mouse models of congenital heart disease to elucidate the pathomechanisms of congenital heart disease. This can lead to potential opportunities that develop therapies and intervention to improve the clinical outcome in patients with congenital heart disease.

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Research

JoVE Journal

Developmental Biology

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

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 ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...