We thank the animal physiology core, division of molecular medicine at UCLA for providing&#160;technical support&#160;and open access to the Vevo 2100 ultrasound biomicroscopy (UBM) system. This study was supported by the NIH/Child Health Research Center (5K12HD034610/K12), the UCLA-Children&#39;s Discovery Institute and Today and Tomorrow Children&#39;s Fund, and David Geffen School of Medicine Research Innovation award to M. Touma.

The University of California, Los Angeles, Animal Care and Use Committee has approved all procedures shown in this protocol. The experiments were conducted as part of an ongoing study under active animal protocols approved by the institutional Animal Care and Use Committee of University of California, Los Angeles, California, USA. Animal handling and care followed the standards of the Guide for the Care and Use of Laboratory Animals.
1. Preparing the High Frequency Ultrasound Imaging System
<ol>
	<li>Turn on the ultrasound imaging system and the physiology monitoring unit.</li>
	<li>Connect the 30/45 MHz transducer.</li>
	<li>Place the corresponding scan head on its holder near the imaging platform.</li>
	<li>Select the Cardiac Measurement Program option.</li>
	<li>Place the ultrasound gel upside down in its pre-warming container set to 37 &#176;C.</li>
	<li>Confirm appropriate tubing system for anesthesia and verify the levels of oxygen and isoflurane.</li>
	<li>Disinfect the imaging platform and the working area.</li>
	<li>Set the heat level of the imaging platform to maintain constant body temperature and heart rate of the dams.</li>
</ol>
2. Pregnant Mouse Preparation
<ol>
	<li>Place the pregnant mouse (C57/BL6) dam in the anesthesia induction chamber.</li>
	<li>Induce anesthesia by using continuously delivered inhalational isoflurane (isoflurane 2%-3%) mixed with 100% oxygen (100% O2) at a flow rate of 200 mL/min in the induction chamber.</li>
	<li>Transfer the sedated animal onto the imaging platform in a supine position.</li>
	<li>Provide steady-state sedation by using a facemask connected to the anesthesia tubing system-delivering isoflurane (1.0% - 1.5%) mixed with 100% O2 at 200 mL/min. 
	CAUTION: Control the leakage of anesthetic gas by using a ventilation system equipped with a charcoal filter containing canister set.</li>
	<li>Tape the limbs gently to the embedded electrocardiographic electrodes after application of the electrode gel to achieve constant monitoring of maternal cardiac and respiratory rates.</li>
	<li>Adjust the level of isoflurane to maintain an average heart rate of (450 +/- 50 beats/min (bpm)).</li>
	<li>Maintain the body temperature within a range of 37.0 &#176;C +/- 0.5 &#176;C. Monitor the body temperature and heart rate being displayed on the physiology controller unit.</li>
	<li>Document the vital signs of the sedated mouse every 15 min throughout the imaging procedure.</li>
	<li>Assess the level of anesthesia by evaluating the mouse&#39;s posture, heart rate, and response to toe pinches.</li>
	<li>Apply ophthalmic balm (1 drop into each eye) to prevent eye dryness and corneal damage.</li>
	<li>Remove the fur from the mid-chest level to the lower limbs by using a depilatory cream to minimize ultrasound attenuation. Remove cream 1-1.5 min after application by alternating wet and dry gauze wipes to prevent damage to the skin.</li>
</ol>
3. Embryo Identification
<ol>
	<li>Palpate the abdominal wall gently to locate the fetuses and spread them out.</li>
	<li>Annotate each embryo on the dam&#39;s abdomen and define their anterior-posterior and dorsal-ventral orientations by using a marker.</li>
	<li>Use the cervix of the sedated dam as a landmark. Label the fetuses on the left and right uterine horns as L1, L2,L 3, etc. (left side) and R1, R2, R3, etc. (right side), respectively (Figure 1A). 
	CAUTION: Avoid spreading the fetuses forcefully. 1-2 fetuses in each litter may overlap with the others, making their positioning and imaging unreliable. Exclude these fetuses from the analysis.</li>
</ol>
4. Fetal Heart Visualization and Annotation
<ol>
	<li>Apply pre-warmed ultrasound gel on the abdomen and spread it carefully to avoid bubble formation. Add additional amount of gel on the area of scan imaging.</li>
	<li>Place the ultrasound probe on its mechanical holder and mobilize it gradually towards the skin to contact with the thick gel layer while looking for the beating heart using the scanning B-Mode (Figure 1).</li>
	<li>Click the scanning B mode button to obtain 2-D images. Use the bladder as a landmark to identify the first fetus positioned in the right or the left uterine horn and mark it as R1 or L1, respectively.</li>
	<li>Confirm the right and left orientation of the individual fetus in real time by moving the imaging platform in the horizontal plane. Scan from head to tail to annotate the snout, the limbs, and the spine as landmarks (Figure 1B, Video 1).</li>
	<li>Visualize the beating heart and annotate the left ventricle (LV) and the right ventricle (RV). Use color Doppler mode to optimize the heart visualization (Figure 1 C-G, Videos 1-2).</li>
	<li>Click the scanning B mode button to obtain a parasternal short axis-view, have the LV and RV displayed in their maximum diameter at the center of data acquisition frame. Start live imaging (Figure 1B-C).</li>
	<li>Change the orientation of the mouse with respect to scanning planes to obtain a longitudinal four-chamber view (Figure 1D). First, identify the remaining structures of the heart such as the atria, interventricular septum, and left and right outflow tracts. Next, have the ventricular and atrial chambers displayed in their maximum diameter. Then start image acquisition.</li>
	<li>Exclude the non-optimal, oblique images from the final analysis. Click the Cini button to obtain continuous recording &#39;Cineloops&#39; for a minimum of 10 s, then save the recorded images.</li>
</ol>
5. Evaluating Fetal Heart Rate and Ventricular Function
<ol>
	<li>Click the scanning M-mode button to obtain cardiac images from four chamber planes (Video 3).</li>
	<li>View the list of the recordings for analysis once the images of all the embryos are complete.</li>
	<li>Exclude the non-optimal, oblique images from the final analysis.</li>
	<li>Click the analysis button to measure wall thickness and the left/right ventricular internal diameter at diastole (LVID, d; RVID, d) and systole (LVID, s; RVID, s), as shown in Figure 2.</li>
	<li>Determine the average fetal heart rate by playing each recorded M-mode tracing and calculating the measurement of one flow cycle to the following flow cycle (the spacing between adjacent peaks).</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain the average heart rate (Figure 2).</li>
	<li>Measure the temporal changes between left ventricular internal diastolic diameter (LVID, d) and left ventricular internal diameter at end systole (LVID, s) throughout the cardiac cycle. Then calculate fractional shortening percentage (FS%) as follows: FS%=[(LVID,d-LVID,s)/LVID,d]x100.</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain the average FS% values.</li>
</ol>
6. Evaluating Cardiopulmonary Flow Parameters
<ol>
	<li>Adjust the sector at an angle of acquisition less than 60o. Click the Doppler Button to perform pulsed wave Doppler measurements from the 2-D four chamber-imaging plane by using a 45-MHz transducer.
	<ol>
		<li>First, visualize the bifurcation of the pulmonary artery to identify the right outflow tract. Next, click pulsed wave Doppler button to obtain the flow pattern through the pulmonary and the aortic valves (Figure 3A, Video 4).</li>
	</ol>
	</li>
	<li>Obtain pulmonary flow measurements from pulsed wave Doppler tracing, including peak systolic velocity (PkV), acceleration time (AT), and ejection time (ET).</li>
	<li>Perform multiple measurements (at least 5 per tracing) to obtain average measurements as shown in Figure 3A (right).</li>
	<li>Calculate the AT/ET ratio for each outflow valve as an indicator of the outflow tracts patency and blood flow.</li>
	<li>Proceed to obtain the mitral and aortic flow patterns from 2-D apical four chamber views using the pulsed wave Doppler. First, identify the left atrial&#160;and left ventricular chambers. Next, place the pulsed wave Doppler sample volume for recording of mitral inflow Doppler patterns and measuring early diastolic velocity ( E) and atrial contraction velocity ( A) (Figure 3B)24,25.</li>
	<li>Adjust Doppler sample volume to obtain the aortic Doppler jet pattern. Use the aortic Doppler jet tracing to measure acceleration time (AT) and ejection time (ET) as shown in Figure 3B (right) (Video 5)</li>
</ol>
7. Evaluating the Feto-Placental Axis
<ol>
	<li>Use the color Doppler scan to visualize the uterine artery and feto-placental vascular tree by using the 45 MHz transducer (Figure 4A).</li>
	<li>Identify the umbilical vessels (two arteries and one vein) in the intra-amniotic segment of the umbilical cord, just after the cord exits from the fetal abdomen.</li>
	<li>Place pulsed wave Doppler sample volume to obtain the umbilical artery flow pattern (Figure 4A).</li>
	<li>Measure vascular peak flow parameters including acceleration time (AT), ejection time (ET), and peak flow velocity at end systole (PkV, s) using the pulsed wave Doppler scanning record (Figure 4B).</li>
	<li>Obtain 5 consecutive waveforms in each vessel, in the absence of fetal movements and&#160;maternal respiratory movements, to measure the average peak velocity for each vessel.</li>
	<li>Proceed to the next embryo.</li>
</ol>
8. Post-Imaging Animal Monitoring
<ol>
	<li>Turn off the isoflurane container following the completion of the imaging process.</li>
	<li>Continue monitoring body temperature, respiratory rate, and heart rate during the recovery phase.</li>
	<li>Remove the facemask and the connected tubing system once the dam starts spontaneous movements.</li>
	<li>Return the dam to the appropriate housing and continue observation according to standard institutional post-procedure protocols.</li>
	<li>Document the time to full resumption of normal activity.</li>
</ol>
9. Performance Requirements and Technical Considerations
<ol>
	<li>Limit the processing time for ~8 fetuses to approximately 1 h to avoid the adverse effects of prolonged anesthesia on vital signs and physiological parameters.</li>
	<li>Complete training with 8-10 pregnant mice to optimize techniques for image acquisition and flow patterns tracing in a short time frame.</li>
</ol>

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No conflict of interest declared.

Cardiovascular malformations and diseases are substantially influenced by genetic factors and environmental elements19. We have previously demonstrated a significant impact of maternal caloric restriction, initiated during the second trimester, on feto-placental circulatory flow and fetal cardiac function9.
Prenatal hypoxia is another common stress factor during fetal development that may tremendously affect the feto-placental physiology and&#160...

Congenital malformations of the heart are heterogeneous structural defects that occur during early cardiac development. Current technical advances of operational procedures have led to significant improvements in the survival rates of&#160;infants with CHDs1,2. However, quality of life is often compromised secondary to&#160;prolonged hospitalization and needs for staged surgical repair procedures1,2,3,4,5. Prenatal detection of the underlying molecular mechanisms of CHDs is crucial in order to plan early interventions, to carry out new prevention strategies, and to improve the lifelong outcomes6,7.
Although multiple genetic and environmental factors have been implicated in CHDs pathogenesis, establishing the causality remains an unmet need to improve diagnostic, therapeutic, and preventive strategies1,8,9,10,11,12. Furthermore, examining the roles of in utero&#160;stress factors and epigenetic modifiers opens new venues for future investigations11,12. The last decade has indeed witnessed rapid advances in next generation sequencing technology including single nucleotide polymorphism (SNP) microarray, whole exome sequencing, and genome-wide methylation studies,&#160;their utilization in studying the genetic causes of complex human diseases, including CHDs1,8,9,10,11&#160;paving the way to identify&#160;novel mutations and genetic variants that have not yet been tested for their pathogenicity in suitable animal models.
Among the different disease model systems, mouse&#160;is the animal model of choice, not only for investigating mechanisms of CHDs during early cardiogenesis13,14,15,16, but also to elucidate their impact on cardiac chamber maturation and function at late gestation in prenatal and perinatal stress factors. Hence, performing in vivo phenotypic characterization of a mutant fetal mouse heart, during both early and late stages of development, is crucial to understand the role of these genetic variations&#160;and environmental factors on&#160;cardiac development, and the potential future impact on chamber specific maturation processes in mice.
Early detection and accurate diagnosis of cardiac defects during development is critical for interventional planning17,18. Being safe, simple, portable and repeatable, fetal sonography has indeed become the standard imaging technique for cardiac evaluation in the clinic. Fetal circulation assessment using Doppler ultrasound has been widely used in clinical practice&#160;not only for the detection of&#160;cardiac defects, but also to detect vascular abnormalities, placenta insufficiency and&#160;intrauterine growth restriction, and to assess the fetal well-being in response to in utero insults including hypoxemia, maternal illness, and drug toxicity17,18. In parallel to its&#160;value in evaluating human defects and diseases, ultrasound assessment of fetal mice has gained increasing utility in experimental settings19,20,21,22,23. In particular, fetal heart ultrasound (echocardiography) allows sequential in vivo visualization of the developing heart. Many experimental studies have used ultrasound-imaging technology to observe fetal cardiovascular development in transgenic fetal mice. Doppler ultrasound has been particularly useful to elucidate the pathophysiological parameters, such as the flow patterns in fetal circulation under physiological challenges or disease conditions10,19. In both humans and animals, abnormal blood flow or oxygen supply to the fetus can result from various conditions that can disrupt fetal environment&#160;in utero&#160;and affect the fetoplacental axis, including placental&#160;abnormalities, maternal hypoxia, gestational diabetes, and pharmaceutically induced vascular constriction15,22. Therefore, establishing standardized methods for performing Doppler ultrasounds on fetal mice will tremendously empower future studies of CHDs by facilitating&#160;monitoring&#160;flow patterns and key hemodynamic indices of the cardiovascular circuits during different stages of cardiac development in genetic mouse models.
High frequency ultrasound has emerged as a powerful tool to measure the developmental&#160;and physiological parameters of the cardiovascular system in mouse models&#160;and human diseases18. This technology has been further refined in recent years. We and other researchers have demonstrated the feasibility of this system for conducting ultra-high frequency ultrasound studies on the fetal mouse heart15,19,20,21,22,23. The system is equipped with Doppler color flow mapping and linear array transducers that generate two-dimensional, dynamic images at high frequency (30 to 50 MHz) frame rates. These advantages, compared to low frequency ultrasound systems and the prior generation of high frequency ultrasound21,22, provide the necessary sensitivity and resolution for in-depth assessment of the fetal circulatory system, including comprehensive characterization of heart structures, chamber function, and flow indices of fetal mice in experimental settings. Herein, we outline methods to perform rapid assessment of cardiopulmonary circulation and feto-placental circulation at embryonic day E18.5 in vivo by using a high frequency&#160;system. We chose a 30/45 MHz transducer that provides an axial resolution of approximately 60 &#181;m and a lateral resolution of 150 &#181;m. However, a higher frequency transducer (40/50 MHz) can be chosen to analyze earlier developmental stages by following a similar methodological approach. The selected M-mode allows the visualization of tissues in motion at high temporal resolution levels (1,000 frames/s). Finally, we&#160;demonstrate the feasibility of high ultrasound for detailed comprehensive phenotypic characterization of fetal cardiovascular hemodynamic status and function in mice at baseline and in response to prenatal hypoxia stress.

<table border="0" cellpadding="0" cellspacing="0" width="1606">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:189px;">Vevo 2100&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:291px;">VisualSonics, Toronto, Ontario, Canada</td>
			<td style="width:301px;">N/A</td>
			<td style="width:827px;">High Freequency Ultrasound Biomicroscopy. The set up is available in animal physiology core facility, division of molecular medicine, UCLA. USA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">inbred mice (c57/BL6)&#160;&#160;</td>
			<td>Charles River Laboratories</td>
			<td>N/A</td>
			<td>Inbread wild type mouse strain</td>
		</tr>
	</tbody>
</table>

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.

Statistical analyses of cardiac and hemodynamic indices were performed offline. The means of 5 consecutive measurements in 3 optimal images were calculated. The data were expressed as Mean &#177; SEM. Student&#39;s t-test was used to infer intergroup comparisons. A P value of &#8804;0.05 was considered statistically significant.
Following the above protocol, we characterized the impact of chronic exposure to pr...

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Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System | Developmental Biology | Video

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.

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 ...

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11.5K Views.  University of California, Los Angeles. 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.

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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

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 ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

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 ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

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 ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...