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) in 1-Day-Old Neonatal Mice6
<ol>
	<li>Separate the 1-day-old neonatal pups from the mother by placing them into a clean cage along with some of the original nesting material.</li>
	<li>Place half of the cage onto a heating pad set to medium heat. The pups should remain on the unheated side of the cage, only being placed onto the heated side after surgery.</li>
	<li>Create a sterile surgical area under an operating microscope. Gather sterilized surgical equipment (Table 1).</li>
	<li>Anesthetize the pup via hypothermia: wrap the pup in gauze to avoid direct skin contact with ice and bury it in an ice bed for approximately 3&#8211;4 min. Check hypothermia of the mice periodically by performing a toe pinch. Neonates tolerate hypothermia well, however, prolonged exposure to hypothermia may result in frostbite and subsequent mortality.</li>
	<li>Once anesthetized, place the mouse onto the surgical area in the supine position, securing the arms and legs with tape. Sterilize the surgical area of the mouse with an antiseptic solution.</li>
	<li>Locate the lower chest region and make a transverse incision in the skin with small dissecting scissors. To widen the surgical view of the ribs, separate the skin from the muscle by lifting the skin gently with a pair of dressing forceps and gently press against the intercostal muscles with the small scissors in the closed position.</li>
	<li>Locate the fourth intercostal space (Figure 1A) and make a small, superficial puncture using sharp forceps, being careful not to puncture any internal organs. Perform a blunt dissection by widening the area in between the intercostal muscles with dressing forceps. Proper anatomical positioning of the incision is essential for appropriate access to the heart.</li>
	<li>Gently guide the heart out of the chest cavity by placing a finger and applying increasing pressure on the left side of the abdomen while holding the intercostal space open with dressing forceps (Figure 1B). Once the heart is outside of the chest, remove the dressing forceps, relieve pressure, and allow the heart to rest on the intercostal muscles.</li>
	<li>Locate the LAD as the area of the heart that has less pooled blood and is in the correct anatomical position (Figure 1C). The LAD can be only seen under the microscope if the heart is accessed within a few minutes of beginning surgery.</li>
	<li>Perform LAD ligation by suturing the LAD with a 6-0 suture (Figure 1D). Tie a square knot twice to induce myocardial infarction (Figure 1E). The depth of the suture into the myocardium may vary, however, proper anatomical positioning of the LAD ligation is crucial for reproducibility. When ligating the LAD, the suture should be pulled tightly but carefully so as not to sever the LAD. Blanching at the apex of the heart will be seen immediately (Figure 1E)</li>
	<li>Allow the heart to slip back into the chest cavity; this process can be gently facilitated with dressing forceps. Suture the ribs together with a surgeon&#39;s knot followed by a 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.</li>
	<li>Remove the tape that was used to secure the hind legs of the pup.</li>
	<li>Adhere the skin together by placing a small amount of skin glue on the upper abdomen. Then, grab the skin of the lower abdomen with fine forceps and cover the exposed chest region. Limit the amount of excess glue that remains on the pups, as this can increase the likelihood of rejection and cannibalism by the mother.</li>
	<li>Immediately facilitate the recovery from anesthesia by placing the pup onto a heating pad set to medium heat. Periodically switch the placement of the neonates to evenly warm all parts of the body.</li>
	<li>Allow the neonate to remain directly on the heating pad for 10&#8211;15 min. Typically, movement is regained within 5 min of being placed onto heat.</li>
	<li>Clean the residual blood and glue with an alcohol wipe.</li>
	<li>Cover foreign scents on neonates by rubbing the entire body with bedding from the original cage. Place the pup into the cage on the heated side while other surgeries are being performed.</li>
	<li>Once all surgeries are completed and pups are warm and mobile, transfer the litter along with the original nesting material to the mother&#39;s cage.</li>
	<li>Monitor the mice for 30&#8211;60 min after surgery and watch for the mother&#39;s acceptance of the pups by nesting and/or grooming.</li>
	<li>Check on the mice the morning following surgery. If mother is distressed and has not nested the pups, consider a foster mother for the pups.</li>
</ol>
2. Clearing the Mouse Heart with Passive CLARITY21,22,23
<ol>
	<li>Anesthetize the mouse with isoflurane. Perform a toe pinch to ensure the mouse is fully sedated.</li>
	<li>Place the mouse onto a clean, surgical area in the supine position, securing the arms and legs with tape.</li>
	<li>Maintain isoflurane sedation on the mouse using a nose cone until the heart is extracted.</li>
	<li>Open the lower chest by holding the fur just below the xiphoid process with tissue forceps and make an incision spanning the width of the ribcage using the large dissecting scissors.</li>
	<li>Cut alongside of the distal portions of the rib cage with surgical scissors.</li>
	<li>Expose the diaphragm muscle by grasping the xiphoid process with tissue forceps. Detach the diaphragm using curved forceps.</li>
	<li>While maintaining a grasp of the xiphoid process, pull the tissue cranially until the beating heart is accessible.</li>
	<li>Grasp the heart at the base with curved forceps and dissect the heart from the chest cavity by cutting the aorta and superior vena cava with iridectomy scissors.</li>
	<li>While the heart is still beating, place the heart into a Petri dish filled with PBS so that the heart pumps out the blood inside as it keeps beating. Myocardial infarction can be confirmed by checking that the placement of the suture is in the proper anatomical position for LAD ligation.</li>
	<li>Gently squeeze the heart with forceps to allow the heart to expel the residual blood.</li>
	<li>Transfer the mouse heart into a disposable 2.5 mL glass shell vial with 2 mL of PBS. Wash away residual blood by incubating the heart on a shaker for 10 min at room temperature (RT) several times. Change the PBS solution each time until PBS remains clear.</li>
	<li>Discard the PBS and fill the vial with 2 mL of cold 4% paraformaldehyde (PFA). Incubate for 4 hours at RT (Figure 2A).</li>
	<li>After incubation, discard the PFA and the vial with 2 mL of PBS. Wash the heart on a shaker for 10 min at RT. Repeat the washing step twice, draining and filling the vial with new PBS each time to fully wash away excess PFA.</li>
	<li>Discard PBS and fill the vial with 2 mL of 4% acrylamide and 0.5% VA-044 solution. Incubate overnight at 4 &#176;C.</li>
	<li>The next day, perform polymerization by transferring the vial to a heat block set at 37 &#176;C for 3 hours.</li>
	<li>Transfer the heart into a new glass shell vial and repeat step 2.12 (PBS wash cycle).</li>
	<li>Discard PBS and fill the vial with 2 mL of Clearing Solution (Table 2). Incubate at 37 &#176;C until the heart is cleared. Change out the solution and refill with fresh Clearing Solution every 2&#8211;3 days. The clearing process could take up to several weeks (Figure 2B-C). 
	NOTE: P1 hearts typically take around 3&#8211;5 days, whereas P21 hearts can take nearly a month before Clearing Solution incubation is complete.</li>
	<li>Discard PBS and fill the vial with 2 mL of PBS and repeat step 2.12 (PBS wash cycle). Refill the vial with fresh PBS and incubate overnight at 37 &#176;C.</li>
	<li>If immunostaining will be performed, skip steps 2.21&#8211;2.22 and proceed to Section 3 for immunostaining. If relying solely on endogenous fluorescence, proceed with steps 2.21&#8211;2.22 and skip Section 3.</li>
	<li>Discard PBS and change the solution to Refractive Index Matching Solution (RIMS) (Table 3). Incubate overnight at 37 &#176;C.</li>
	<li>After incubation, the cleared tissue can be stored in RIMS solution at RT. Tissue may appear to be white and opaque in the center when first transferred into RIMS; tissue should become transparent after being incubated in RIMS at room temperature for several weeks (Figure 2D).</li>
</ol>
3. Optional: Immunohistochemistry Staining of the Whole-Mount Mouse Heart
<ol>
	<li>Remove the cleared heart from the RIMS solution and place into a clean 2.5 mL glass vial with 2 mL of PBST (PBS with 0.1% Triton-X 100)</li>
	<li>Wash the heart in PBST 3 times on an orbital rotator with 30 min incubations at RT.</li>
	<li>Block non-specific antibody binding by immersing the heart in 2 mL of 20% blocking buffer (diluted in PBST) and incubate with rotation for 3 hours at RT. Transfer to 4 &#176;C to stain with rotation overnight. 
	NOTE: Blocking buffer is made from normal serum matching the species in which the secondary antibody was raised.</li>
	<li>Wash the heart in PBST 3 times with rotation for 5 min incubations at RT.</li>
	<li>Immerse the heart in primary antibody (diluted in 2% blocking buffer with PBST) and prevent light exposure by wrapping the glass vial in aluminum foil. Incubate with rotation overnight at RT. 
	NOTE: From this point forward, aluminum foil should be continuously used to protect the secondary from ambient light exposure.</li>
	<li>Incubate for an additional 24 hours with rotation at 4 &#176;C.</li>
	<li>Following primary staining, repeat step 3.2 (long PBST wash cycle) to remove the unbound primary antibody from the tissue. Extend the wash with an overnight incubation with rotation at RT.</li>
	<li>Working in an area with limited lighting to prevent secondary antibody light exposure, immerse the heart in secondary antibody (diluted in 2% blocking buffer with PBST) and incubate with rotation for 3 hours at RT. Transfer to 4 &#176;C to stain with rotation overnight.</li>
	<li>On the next day, repeat step 3.2 (long PBST wash cycle) to remove unbound secondary antibody.</li>
	<li>Replace the solution with 2% blocking buffer (diluted in PBST). Remove residual unbound antibody by washing overnight with rotation at RT.</li>
	<li>The next day, check that excess secondary antibody has been removed using confocal microscopy. Extend the wash as needed, replacing the 2% blocking buffer solution daily. Proceed once little to no non-specific secondary is present.</li>
	<li>Store the immunostained heart in PBS at 4 &#176;C.</li>
	<li>Directly before whole-mount microscopy, incubate the heart in RIMS solution overnight at 37 &#176;C. Extend the incubation an additional 24 hours if the tissue is still not fully cleared.</li>
	<li>Store the fully cleared and immunostained heart in RIMS at RT.</li>
</ol>
4. Visualizing Non-myocyte Populations in 3D with Single-Photon Confocal Microscopy Imaging of the Cleared Mouse Heart
NOTE: If mouse hearts are harvested embryonically, continue with section 4.1. For mouse hearts harvested postnatally, continue with section 4.2.
<ol>
	<li>Imaging the Cleared Embryonic Mouse Heart
	<ol>
		<li>Fill the microscope depression slide with the PBS solution.</li>
		<li>Carefully pick up the cleared heart with curved forceps and place the tissue onto the slide.</li>
		<li>Mount the slide with a glass coverslip. The tissue can now be imaged under a confocal microscope.</li>
	</ol>
	</li>
	<li>Imaging the Cleared Postnatal Mouse Heart
	<ol>
		<li>Fill half of the chamber of the depression slide with PBS solution. In order to create a chamber large enough to fit an adult mouse heart, a 3D-printed polypropylene depression slide was custom-made (Figure 4).</li>
		<li>Carefully pick up the cleared heart with curved forceps and place the tissue into the chamber. Fill the remaining volume of the chamber with PBS.</li>
		<li>Fill the chamber with PBS so the surface of the liquid forms a dome above the top of the chamber. Mount the cover slide. The tissue can now be imaged under an upright confocal microscope.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

5.9K Views.  University of Wisconsin-Madison School of Medicine and Public Health. 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.

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

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, ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Medicine

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Bioengineering

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...