Name: capturing the cardiac injury response of targeted cell populations via cleared heart three-dimensional imaging
Uploaded: 2020-03-17T13:00:00
Description: Watch this Scientific Journal Video about Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging at JoVE.com

Funding for this project was provided by the UW School of Medicine and Public Health from the Wisconsin Partnership Program (A.I.M.), and an American Heart Association Career Development Award 19CDA34660169 (A.I.M.).

All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and in compliance with the Institutional Animal Care and Use Committee in the School of Medicine and Public Health at the University of Wisconsin&#8211;Madison. All methods were performed on wild type C57BL/6J (B6) and transgenic mouse lines obtained from Jackson Laboratories.
1. Coronary Artery Occlusion (Myocardial Infarction) Induced via Ligation of the Left Anterior Descending Artery (LAD) ...

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Cell-cell interactions between cardiomyocytes and non-myocyte populations are a determining factor of whether the heart will undergo fibrosis or repair following injury. Discoveries have been made demonstrating that a variety of cell types, including nerves14, epicardial cells24, peritoneal macrophages25, arterioles12,13, and lymphatic endothelial cells26, all play an ...

The heart has long been considered to be a post-mitotic organ, yet recent evidence demonstrates that cardiomyocyte renewal occurs in the adult human heart at about 1% per year1. However, these low rates of cardiomyocyte turnover are insufficient to replenish the massive loss of tissue that occurs following injury. A heart that has suffered a myocardial infarction will lose around one billion cardiomyocytes, often serving as a prelude to heart failure and sudden cardiac death2,3. With over 26 million people affected by heart failure worldwide, there is an unmet need for therapeutics that can reverse the damages inflicted by heart disease4.
In order to bridge this gap in therapeutics, scientists have begun investigating evolutionarily conserved mechanisms that underlie endogenous regeneration following injury. One model for studying mammalian cardiac regeneration is the neonatal mouse. Within the week following birth, neonatal mice have a robust regenerative response following cardiac damage5. We have previously demonstrated that neonatal mice can regenerate their heart via cardiomyocyte proliferation following an apical resection5. Although this technique can evoke cardiac regeneration in the neonates, the surgery lacks clinical relevance to human heart injuries. In order to mimic a human injury in the neonatal mouse model, we have developed a technique to induce a myocardial infarction through a coronary artery occlusion6. This technique requires surgical ligation of the left anterior descending artery (LAD), which is responsible for delivering 40%&#8211;50% of the blood to the left ventricular myocardium6,7. Thus, the surgery results in an infarct that impacts a significant portion of the left ventricular wall. This damage to the myocardium will stimulate cardiomyocyte proliferation and heart regeneration in neonates5.
The coronary artery occlusion surgery provides a highly reproducible and directly translational method to uncover the inner workings of cardiac regeneration. The neonatal surgery parallels coronary artery atherosclerosis in the human heart, where accumulation of plaque within the inner walls of the arteries can cause an occlusion and subsequent myocardial infarction8. Due to a void in therapeutic treatments for heart failure patients, an occlusion in the LAD is associated with mortality rates reaching up to 26% within a year following injury9, and consequently has been termed the &#34;widow maker.&#34; Advancements in therapeutics require a model that accurately reflects the complex physiological and pathological effects of cardiac injury. Our surgical protocol for neonatal mouse cardiac injury provides a platform that allows researchers to investigate the molecular and cellular cues that signal mammalian heart regeneration after injury.
Recent research highlights the dynamic relationship between the extracellular environment and proliferating cardiomyocytes. For example, the postnatal regenerative window can be extended by decreasing the stiffness of the extracellular matrix surrounding the heart10. Biomaterials from the neonatal extracellular matrix can also promote heart regeneration in adult mammalian hearts following cardiac injury11. Also accompanying cardiomyocyte proliferation is an angiogenic response12,13; collateral artery formation unique to the regenerating heart of the neonatal mouse was shown to be essential for stimulating cardiac regeneration12. Moreover, our lab has demonstrated that nerve signaling regulates cardiomyocyte proliferation and heart regeneration via modulation of growth factor levels, as well as the inflammatory response following injury14. These findings emphasize the need to trace non-myocyte cell populations in response to cardiac injury. In order to accomplish this goal, we have taken advantage of the Cre-lox recombination system in transgenic mice lines to incorporate constitutive or conditional expression of fluorescent reporter proteins for lineage tracing. Furthermore, we can use advanced methods to determine clonal expansion patterning with the Rainbow mouse line, which relies on stochastic expression of the Cre-dependent, multi-color fluorescent reporters to determine the clonal expansion of targeted cell populations15. Employing lineage tracing with the neonatal coronary artery occlusion surgery is a powerful tool for dissecting the intricate cellular mechanisms of cardiac regeneration.
Tracking the lineage of fluorescently labeled cells with three-dimensional (3D) whole organ imaging is difficult to achieve using traditional sectioning and reconstruction technique &#8211; especially when cell populations are fragile, such as nerve fibers or blood vessels. While direct whole-mount imaging of the organ by optical sectioning can capture superficial cell populations, structures that reside deep within the tissue remain inaccessible. To circumvent these barriers, tissue clearing techniques have been developed to reduce the opacity of whole organ tissues. Recently, significant advances have been made to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel (CLARITY)&#8211;based methods, which clear fixed tissue via lipid extraction16. Steps are also taken to homogenize the refractive index and subsequently reduce light scattering while imaging17. One such method is active CLARITY, which expedites lipid decomposition by using electrophoresis to penetrate the detergent throughout the tissue18. Although effective, this tissue clearing method requires expensive equipment and can cause tissue damage, making the approach incompatible with fragile cell populations such as the cardiac nerves19. Thus, we employ the passive CLARITY approach, which relies on heat to gently facilitate detergent penetration, therefore aiding in the retention of intricate cell structures20,21.
Passive CLARITY is typically thought to be less efficient than active CLARITY18, as the technique is often accompanied by two major obstacles: the inability to clear the entire organ depth and the extensive amount of time required to clear adult tissues. Our passive CLARITY approach overcomes both of these barriers with an expeditated clearing process that is capable of fully clearing neonatal and adult heart tissue. Our passive CLARITY tissue clearing technique has reached an efficiency that permits the visualization of a variety of cardiac cell populations, including rare populations distributed throughout the adult heart. When the cleared heart is imaged with confocal microscopy, the architecture of cell-specific patterning during development, disease, and regeneration can be illuminated.

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			<td>1-thioglycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Prolene Sutures</td>
			<td>Ethicon</td>
			<td>8889H</td>
			<td>Polypropylene Sutures</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Excelta</td>
			<td>16-050-146</td>
			<td>Half Curved, Serrated, 4 in</td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>Fisherbrand</td>
			<td>13-812-39</td>
			<td>Dissecting, 4.5 in</td>
		</tr>
		<tr>
			<td>Glass Vial</td>
			<td>Fisherbrand</td>
			<td>03-339-26A</td>
			<td>12 x 35 mm Vial with Cap</td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Density gradient medium</td>
		</tr>
		<tr>
			<td>Iridectomy Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td>2 mm Cutting Edge</td>
		</tr>
		<tr>
			<td>Large Dissecting Scissors</td>
			<td>Fisherbrand</td>
			<td>08-951-20</td>
			<td>Straight, 6 in</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Fisherbrand</td>
			<td>08-966</td>
			<td>Mayo-Hegar, 6 in</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp Forceps</td>
			<td>Sigma-Adrich</td>
			<td>Z168777</td>
			<td>Fine Tip, Straight, 4.25 in</td>
		</tr>
		<tr>
			<td>Small Dissecting Scissor</td>
			<td>Walter Stern Inc</td>
			<td>25870-002</td>
			<td>30 mm Cutting Edge</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps</td>
			<td>Excelta</td>
			<td>16050133</td>
			<td>Medium Tissue, 1X2 Teeth</td>
		</tr>
		<tr>
			<td>VA-044</td>
			<td>Wako Chemicals</td>
			<td></td>
			<td>Water-soluble azo initiator</td>
		</tr>
	</tbody>
</table>

capturing the cardiac injury response of targeted cell populations via cleared heart three-dimensional imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Often the two most challenging steps are guiding the heart out of the chest cavity and ligating the LAD. To troubleshoot these steps, adjustments may be made in the placement of the initial puncture between the fourth intercostal muscles; if the puncture and blunt dissection are too close in proximity to the sternum, the heart may not be able to exit the chest cavity (Figure 1A).
Additionally, increased pressure on the left abdomen may be needed t...

Watch this Scientific Journal Video about Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging at JoVE.com

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

The endogenous heart regeneration is a dynamic process that involves multiple cell types working together in concert to mount a robust regenerative response. Peering into the regenerating tissue via 3D imaging of the cleared heart in response to injury can aid in reconstruction of the cellular dynamics that guide this cardiac regenerative response. Our protocol is specifically designed to track cells during development and regeneration.We foresee no limitations in broadly applying our clearing, staining and imaging techniques to other organ types. For LAD-induced coronary artery occlusion, first sterilize the surgical area of an anesthetized one-day-old neonatal pup with antiseptic solution. Make a transverse skin incision in the chest region.To separate the skin from the muscle, use a pair of dressing forceps to lift the skin carefully while gently pressing the scissors against the intercostal muscles in a closed position. Locate the fourth intercostal space and use small forceps to make a small superficial puncture taking care to avoid the internal organs. Using blunt dissection and the dressing forceps, widen the area between the intercostal muscles just until a finger can be applied to the left side of the abdomen while holding the intercostal space open with dressing forceps to gently guide the heart out of the chest cavity.Once the heart has been extracted, allow the heart to rest on the intercostal muscles and locate the LAD as the region of the heart with less pooled blood. To induce myocardial infarction, use a 6-0 suture to tie a square knot tightly around the vessel two times without severing the artery. Blanching at the apex should be observed immediately.It's important to remain consistent on the suture placement along the LAD for a similar infarct size. This is best achieved by accessing the heart quickly while the blood is still flowing through the arteries. When the suture has been placed, allow the heart to slip back into the chest cavity.Suture the ribs together with one surgeon and one square knot using blunt forceps to lift the upper set of ribs while passing a 6-0 suture through the upper and lower set of ribs. Apply a small volume of skin glue to the upper abdomen and use fine forceps to position the skin of the lower abdomen over the exposed chest region. Then place the pup on a heating pad and perform the ligation on the next animal.When all of the surgeries have been performed, return the warm and mobile pups to the mother's home cage. For passive clarity of the harvested heart tissue, 21 days postsurgery after confirming a lack of response to toe pinch, place the mouse onto a clean surgical area in the supine position and secure the limbs with tape. Using tissue forceps, grasp the fur just below the xiphoid process and make an incision spanning the width of the ribcage.Cut alongside the distal portions of the ribcage and use the forceps to lift the xiphoid process to expose the diaphragm. Detach the diaphragm with curved forceps and pull the tissue cranially until the beating heart is accessible. Use the curved forceps to grasp the heart at the base and use iridectomy scissors to cut the aorta and superior vena cava.Place the beating heart into a Petri dish of PBS so that the heart pumps out any retained blood. The myocardial infarction can be confirmed by a correct anatomical placement of the LAD ligation. When the heart stops beating, gently squeeze the organ with forceps to expel the residual blood and transfer the specimen into a disposable 2.5 milliliter glass shell vial containing two milliliters of PBS.Place the vial onto a shaker for at least 30 minutes at room temperature changing the PBS solution every 10 minutes until the solution remains clear of blood. When the PBS is clear, replace the PBS with two milliliters of cold 4%paraformaldehyde for a four-hour incubation at room temperature followed by three 10-minute washes on the shaker with two milliliters of fresh PBS per wash. After the last wash, fill the vial with two milliliters of 4%acrylamide supplemented with 0.5%VA-044 solution for an overnight incubation at four degrees Celsius.The next day, place the vial in a 37 degrees Celsius heat block for three hours. At the end of the polymerization, transfer the heart into a new glass shell vial and wash the heart tissue in two milliliters of fresh PBS 10 minutes at a time refreshing the PBS at the end of every incubation. When the PBS is clear, replace the wash buffer with two milliliters of clearing solution.Incubate the specimen at 37 degrees Celsius for several weeks until the heart is clear, refreshing the clearing solution every two to three days. When the organ is clear, wash the specimen with PBS on the shaker as demonstrated before refilling the vial with fresh PBS for a 24-hour incubation at 37 degrees Celsius. The next day, replace the PBS with RIMS for a 24-hour incubation at 37 degrees Celsius.At the end of the incubation, the tissue is fully cleared. The tissue should become transparent after several weeks. For imaging of the cleared postnatal mouse heart after myocardial infarction induction, fill half of a chamber of a custom-made depression slide with PBS and use curved forceps to carefully transfer the cleared heart into the chamber.Fill the remaining volume of the chamber with PBS. Then fill the chamber with PBS until the surface of the liquid forms a dome above the top of the chamber and mount the cover slide before imaging the tissue by confocal microscopy according to standard imaging protocols. If the puncture and blunt dissection are too close in proximity to the sternum, the heart may not be able to exit the chest cavity.If the heart does not easily exit the cavity, apply pressure to the left abdomen to facilitate the process. Complications may occur from resting the heart on the intercostal muscles. Therefore, it is important to keep the blunt dissection to a minimal size in a horizontal orientation to allow for a clear visualization and accessibility of the LAD.Note that a superficial ligation will have less room for adjustment in the final suture placement and that the suture tying around the LAD should be performed with controlled steady movements. By the end of the clearing step, the entire heart should be consistently opaque with no discoloration in the center. After a few days in RIMS solution at room temperature, the heart should be completely cleared and some tissue expansion may be observed.As observed by tdTomato reporter protein expression, the cardiac nerves are mainly superficial with some populations residing in the epicardial layer. Importantly, reporter protein expression confirmation is preserved after undergoing the clarity protocol as demonstrated. Clearing and imaging the 3D heart provides perspective into the location and morphology of cardiac cell populations.This allows us to identify new aspects in cell patterning and cell-cell interactions.

capturing-cardiac-injury-response-targeted-cell-populations-via

Research

JoVE Journal

Developmental Biology

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...