A modular platform to generate functional sympathetic neuron-innervated heart assembloids

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.


Introduction
Human pluripotent stem cell (hPSC)-based 3D organoid strategies have become powerful tools to study human organ development, for disease modeling and drug discovery over the past years.Assembloids result from the integration of multiple organoids or a combination of organoids with other cell types [1][2][3] .
Most organs of the body, except the brain, are innervated and thus regulated by the autonomic nervous system.Speci cally, the sympathetic nervous system is responsible for activation of cells in most organs.
A key challenge in the organoid eld is the lack of innervation of organoids, despite neural regulation being vital for organ and thus organoid development, integrity, and function.To address this, we present a simple, easy to reproduce and cost-effective method without the need for bioengineering or special instrumentation to create sympathetic neuron (symN)-innervated cardiac organoids.The modular approach allows adaptation for innervation of various organoid types, like lung, kidney, or liver, by using symNs; or for innervation of cardiac organoids by other neuron types, such as parasympathetic or sensory neurons.
Innervation of peripheral tissue organoids has been reported.Intestinal organoids were innervated via mixing of vagal neural crest cells into human intestinal organoids (HIOs) 9 .This elegant study resulted in organoids containing functional enteric neurons and glia.However, the enteric nervous system is speci c to the gastrointestinal tract and thus cannot easily be adapted for innervation of other organ type organoids, thus it lacks versatility.Neuromesodermal progenitor (NMP)-derived organoids were reported, where the NMP gave rise to both skeletal muscle and motor neurons from the same progenitor within the same cultures 10 , as an one-pot differentiation 11 .This exciting study, however, is not easily adaptable to other organ type organoids, if a common neural-tissue speci c progenitor is not readily available.For example, it would be challenging to differentiate symN-innervated lung organoids, since there are no common lung and symN progenitors known.Also, one-pot differentiation protocols make it di cult to perform cell type-speci c, genetic manipulations within the organoids.Schneider et al., showed exciting results of bioengineered heart organoids with assembled autonomic organoids creating an innervated cardiac assembloid 12 .This elegant study requires advanced bioengineering capabilities and isometric force instrumentation for analysis 12 .Such approaches are not easily accessible to many researchers.We describe a simple, highly versatile strategy for sympathetic innervation of cardiac organoids, that may be adapted to most organ's organoid types and to other neuron types by other researchers.
Cardiovascular disease affects millions of people worldwide, and costs billions to the health care systems 13 .The most well-known and life-threatening cardiovascular diseases, such as heart failure and heart attack, may happen when blood supply is not regulated properly 13 .In addition to genetic heart defects, risk factors for heart failure and heart attack include metabolic conditions, such as diabetes and obesity, hypertension, viral infection, drug abuse, smoking, and excessive alcohol intake 13 .Interestingly, many of these risk factors are mediated by the sympathetic nervous system (SNS).The SNS belongs to the autonomic nervous system and regulates various cardiovascular functions, including heart rate, blood pressure, blood glucose, and gland secretion 14 .SymN hyperactivity and neuropathy have been associated with the pathology of diabetes and obesity, hypertension, viral infection, drug abuse, smoking, and alcohol toxicity in adults [15][16][17][18][19][20] .Furthermore, symNs innervate the heart as early as the embryonic stage (about E13.5), which may participate in hyperplastic to hypertrophic transition of the heart at neonatal stage and cardiac maturation [21][22][23] .Current studies have shown that developmental disorders that lead to aberrant symN innervation to the heart will cause heart rate variability and arrythmia, which increase the risk of sudden cardiac death [24][25][26][27] .
Here, we developed a novel, easy to reproduce and highly versatile neurocardiac organoid system using the assembloid technology, which refers to the assembly of more than two pre-differentiated cell types into an organoid structure 1,2,28 .The assembloid strategy has recently become popular, since it enables (i) the modeling of complex organs, (ii) modeling of interactions between various organs, or (iii) modeling of different regions in the body (for example in the brain) 1,2,28 .Given that we have recently established a chemically-de ned, well-characterized protocol to differentiate highly pure symNs from hPSC 29,30 , and that there are many well-established CMs or cardiac organoid differentiation protocols available 31,32 , we sought to build a sympathetic-cardiac organoid as an assembloid.Herein, we combined early symN progenitors with early cardiac progenitors to form assembloids, named human sympathetic cardiac assembloids (hSCAs).The process of hSCA assembly is in free oating 3D culture, which is relatively easy to work with and reproducible by many stem cell labs.Cardiac tissues in our hSCAs were innervated by symNs and showed structural and functional maturation.SymNs in the organoids were functional and were able to regulate cardiac contraction and maturation.Hypoxic stress induced endogenous NE over secretion, and synergistically led to cardiac infarction features in hSCAs.This symN-heart axis assembloid paradigm therefore provides not just an ideal platform for assessing the mechanisms that environmental factors exert on cardiac function through sympathetic regulation, but also a playbook for generating innervated organoids from other organ systems.
The heart consists of multiple cardiac lineages, including smooth muscle cells (of the vasculatures), cardiac epithelial and endothelial cells, and cardiac broblasts that produce the extracellular matrix 33,34 .
For our hSCAs, we chose a CM differentiation protocol modi ed from Lin et al., 35,36 to generate early cardiac progenitors, which may still have the multipotency to be differentiated into several of the cardiac lineages 37,38 .hPSCs are cultured in 2D, and rst induced by the WNT activator CHIR99021, followed by WNT inhibition using XAV939.Cardiac progenitors expressed typical markers, including GATA4, WT1, ISLET-1, and CD56 shown by RTqPCR on day 7 (Fig. 1c).To form hSCAs, dissociated symNblasts (day 14) and cardiac progenitors (day 7) were mixed and cultured in low attachment plates, in oating cultures on a shaker for up to 5 weeks (Fig. 1d).The size of the hSCAs increased until week 3 and stabilized after (Fig. 1d-e), which was in line with decreased Ki67 expression from wk1 to wk5 (Fig. 1f).The hSCAs were beating spontaneously with a beating rate that increased over time (Fig. 1g), which we quanti ed via video-based image quanti cation (Suppl.Figure 1, movie S1).These results suggest that on wk5, hSCAs have stopped proliferating and may have entered their maturation stage.

hSCAs self-organization
One of the gold standards for a quali ed organoid/assembloid is its ability of cellular and structural selforganization, often indicating maturation and functionality 40 .Therefore, we rst evaluated the maturity of the cardiac tissue within the hSCAs on wk5 via the ratio of MYH7/6 and MYL2/7 expression, which indicate human cardiac tissue maturity if they are above 1 41 .In wk5 hSCAs, both MYH7 and MYL2 levels were higher than MYH6 and MYL7, respectively (Fig. 2a).To further examine the hSCAs maturity and organization, we assessed wk5 hSCAs by transmission electron microscopy (TEM).We observed the ultra-structures of well-aligned myo ber bundles, Z-lines, and intercalated discs (Fig. 2b).In addition, transverse tubules (t-tubules) are internalized CM membranes that surround the muscle bers and are enriched in ion channels, which are crucial for mature excitation-contraction coupling and heart function 42,43 .RT-qPCR analysis of wk5 hSCAs identi ed the expressions of T-tubule markers RYR2 and CAVEOLIN3 (Fig. 2c).Accordingly, we detected T-tubule protein structures within the cardiac tissues using TEM (Fig. 2d), and via wheat germ agglutinin (WGA) staining (Fig. 2e).
Interestingly, in about 50% of hSCAs in each differentiation (Fig. 2f), we observed cavity structures (Fig. 2g).Cavities were compartmentalized by a single layer of cells, excluding the possibility that the cavities are caused by necrosis that is often seen in long-term cultured organoids 44 .Similarly, a single cell layer was also formed in the exterior of wk5 hSCAs (Fig. 2g).To assess whether the cavity structures are mimicking heart chambers, we stained hSCAs for epicardium marker WT1 and endocardium marker NFATC1.The results showed that the exterior cell layer was WT1 + , while the interior layer was TFATC1 + (Fig. 2h), suggesting both epicardium and endocardium patterning within the hSCAs.Furthermore, hSCAs showed polarized patterning for the atrial marker MLC-2a and ventricular marker MLC-2v (Fig. 2i).Using microelectrode array (MEA), we detected a propagating beating pattern in hSCAs (Fig. 2j).Together, our data suggest that wk5 hSCAs are self-organized in their structure and cell type variety, are relatively mature, and the tissues are functional.

Functional coupling and sympathetic regulation in hSCAs
Next, we asked whether the symNs in hSCAs are physically innervating the heart muscles.Using light sheet microscopy, we reconstructed the 3D muscle mass in hSCAs in high resolution (movie S2).Using whole mount staining, we observed that symNs were deeply associated with and growing throughout the cardiac tissues (Fig. 3a).The points of the physical contact between symN axons and cardiac tissues, which form classic swelling and nodal structures 45,46 , were also identi ed (Fig. 3a, arrows).We then stained the symN axon terminals with VMAT2 and TH and con rmed the co-localized signals of both within the cardiac tissue, as well as the nodal bouton en passant-like structure along the axon terminals (Fig. 3b, white arrows).Accordingly, TEM imaging also con rmed the physical innervation of symN axons to CMs in hSCAs (Fig. 3c).
Aside from the physical connection, we examined whether symNs in the assembloids are able to regulate cardiac function.NE is the main neurotransmitter used by symNs and is critical for functional regulation of the heart 20,47 .Thus, we sought to examine if symNs in hSCAs synthesize and release NE.NS510 is a highly sensitive NE chemical probe that has been used to study real-time NE synthesis and dynamics in chroma n cells and symNs 29,48 .Using NS510, we detected NE throughout the assembloids (Fig. 3d).The NE level was also detectable in cell lysates by ELISA (Fig. 3e).To assess symN activity, we performed calcium (Ca 2+ ) imaging using Fluo-4 Ca 2+ labeling.To be able to distinguish Ca 2+ uxes in symNs and not in cardiac tissues, we differentiated symNblast using the EF1-RFP reporter hPSC line and mixed them with unlabeled cardiac progenitors (Fig. 3f).Over time, we observed Ca 2+ sparks (green) rst in symNs (red, Fig. 3f), followed by sparks in cardiac cells, which are downstream of the axons of the symNs (Fig. 3f).This result demonstrates that both symNs and cardiac tissues in hSCAs are functional, and cardiac activity can be regulated by symN activation.To test if we can manipulate hSCA beating by activating symNs, we used two methods to stimulate symNs within the hSCAs: (1) pharmacological activation using nicotine (1 µM), a widely used method in in vitro symN and CM cocultures that has been shown to exclusively activate symNs in short-term treatment 29,[49][50][51] .(2) We differentiated symNblasts from an optogenetic iPSC line that expresses ChR2, and can be activated by blue light exposure 52 .Both nicotine treatment and blue light exposure resulted in increased hSCA beating e ciency (Fig. 3g).Together, our data suggest that symNs and cardiac tissues in hSCAs are functionally connected, and that the cardiac tissue can be manipulated by symN activation.

SymNs regulate cardiac development through NE signaling
The effect of SymN activation on heart development has been reported, potentially through NE and adrenergic signaling 22,23 .In vitro 2D co-cultures using hPSC-derived or primary cultured symNs and CMs also demonstrated that symN connectivity facilitates CM maturation 53 .We re-analyzed bulk RNA seq.data comparing hPSC-derived symNBlasts (SSRN: https://dx.doi.org/10.2139/ssrn.4318816) and symNs 29 , and found that during maturation of the neurons alone (Fig. 1a), GO terms of genes that are involved in cardiac development and regulation pathways were signi cantly upregulated (Fig. 4a), suggesting that the neurons acquire the capability to regulate and mature cardiac tissue.To test if the symNs in our hSCAs play a regulatory role on cardiac development and maturation, we treated the assembloids with α-and β-adrenergic receptor antagonist labetalol (LAB, 1 µM) to fully block the NE signaling during the growth of the hSCAs (Fig. 4b).The drug was given from wk3, the stage when the assembloid growth almost reaches its plateau (Fig. 1e).Given that symN activity may promote cellular hyperplastic to hypertrophic transition of CM 22,23 , we rst compared the overall size of hSCAs on wk5 after LAB treatment.We did not observe differences in assembloid size between DMSO or LAB treated hSCAs (Fig. 4c).However, RT-qPCR analysis for markers of CM maturity 53,54 (CX43, α-ACTININ, and CD36) revealed decreased expressions upon LAB treatment (Fig. 4d).This result suggests that symNs promotes cardiac development through NE signaling in hSCAs; however, NE signaling alone may not be responsible for cardiac hypertrophic transition.

hSCAs model hypoxia-induced cardiac infarction through endogenous NE
To test the effect of sympathetic input to cardiac function in the assembloids in a diseased state, we sought to model myocardial infarction.Richards et al. has established an elegant model of myocardial infarction in their cardiac organoids 55 .In this model, a moderate hypoxic environment (10% O 2 ) was applied, which prevented massive and sudden cell death in 3D cardiac microtissues and allowed the observation of the progression of infarction.In addition to hypoxia, exogenous NE was added to the cardiac organoids to mimic the effects of increased sympathetic tone to the heart.The combined treatments resulted in infarction of the cardiac organoids at a state similar to mice with myocardial injury 55 .Here, we took advantage of this infraction model and applied their conditions to our hSCAs.We subjected hSCAs to 10% O 2 for 10 days without exogenous NE and examined whether the symNs in the organoids become responsive to the hypoxic stress (Fig. 5a).After low oxygen treatment, hypoxic hSCAs were recognized by Image-iT™ Hypoxia Reagent compared to normoxic controls (Fig. 5b).The hypoxic stress stimulated overproduction of endogenous NE from symNs in hSCAs, measured via the NS510 probe and ELISA (Fig. 5c).Extracellular matrix in the heart, such as collagen, supports heart structural organization and development, but is also the component that forms the scar tissue in a damaged heart [56][57][58] .TEM imaging identi ed collagen in hSCAs (Fig. 5d).It has been shown that aberrant ECM accumulation and imbalanced degradation in the heart leads to the increased stiffness in cardiac brosis [56][57][58] .To evaluate this stiffening effect in our cardiac infarction mimicking hSCAs, we used atomic force microscopy (AFM) to measure the stiffness and compare it between normoxia or hypoxiatreated hSCAs (Fig. 5e and Suppl.Figure 2).As expected, hypoxic hSCAs showed increased stiffness compared to hSCAs in the normoxic environment (Fig. 5e).To further support the ndings of brosis, hSCAs were stained for cleaved caspase-3 (c-Cas3) to detect apoptotic cells in the organoids.Compared to normoxic controls, hypoxic hSCAs displayed high amounts of c-Cas3 + cells in the center of the assembloids, likely due to the de ciency in oxygen supply (Fig. 5f).In addition, using the cardiac broblast marker vimentin that labels brotic tissue, we observed increased vimentin signals on the outskirt of the hypoxic hSCAs.Vimentin + cells in that area showed elongated and elastic morphology, which is a typical feature of brotic tissues 55 (Fig. 5f).We further con rmed the brotic cardiac scar tissues in hypoxic hSCAs by assessing the colocalizing level of α-SMA and F-actin 55 .In the outskirt area, hypoxic hSCAs showed higher colocalization of α-SMA + /F-actin + cells compared to normoxic hSCAs (Fig. 5f).RT-qPCR analysis showed that the expression of Ca 2+ handling genes 55 were altered in hypoxic hSCAs, with a pattern similar to what Richards et al. showed, suggesting that Ca 2+ handling capacity was impaired in hypoxic hSCAs (Fig. 5g).hSCAs present a powerful tool to screen drugs for heart failure.In the clinic, β-blockers (propranolol, a βAR antagonist), which blocks the excitatory effects of NE to the heart are prescribed for heart failure and to prevent a second infarction 59,60 .Thus, to further evaluate the potential of hSCAs for future cardiotoxicity studies, we treated hypoxic hSCAs with propranolol (Fig. 5a).
When treating hSCAs with propranolol (1 µM) in addition to low oxygen, the impaired expression of Ca 2+ handling genes were rescued (Fig. 5f).These results suggest that hSCAs possess a functional symNcardiac tissue axis, which is responsive to environmental inputs at both healthy and diseased states and may be used for cardiotoxicity screening.

Discussion
Organoids are self-organizing 3D cell cultures that mimic some of the cellular, structural, and functional complexity of the native organ in vitro.They provide valuable insights to understand the structurefunction relationship of human organs that 2D models cannot achieve, such as brain lobe structure, renal pyramid function and heart spatial patterning.Assembloids result from the integration of multiple organoids or combination of organoids with other cell types [1][2][3] .They have the advantage of enabling the study of interaction of tissues that may not normally develop from the same progenitor, for example forebrain and hindbrain 1 or vasculature and brain tissue 61 .A major outstanding issue in the organoid/assembloid eld is the lack of innervation of peripheral tissue organoids.This is despite the fact, that almost all organs outside the brain are innervated by the peripheral nervous system, and that this neural regulation is crucial to the development, integrity, and function of organs.
Few innervated organoids have been reported to date; however, the common disadvantage of those exciting reports is that they cannot be easily adapted to other organ type organoids or are technically di cult to reproduce by researchers.Workman et al. mixed speci ed hPSC-derived vagal NC cells with developing gut tube organoids and created an intestinal organoid with enteric neuron innervation.This elegant work laid the conceptual groundwork for innervating peripheral tissue organoids 9 .However, enteric neurons are speci c to the GI tract and do not innervate other organs.Thus, making this less universal as an innervation strategy for many different organoid types.Schneider et al. created a 3D bioengineered hPSC-derived CM model with autonomic innervation using hPSC-derived autonomic organoids 12 .CMs in this model were plated in circular form on dynamic stretch devices, which achieved advanced maturation of cardiac tissues.Autonomic neural organoids were then stuck into the ring of the engineered cardiac organoids for innervation and regulation thereof.However, these assembloids did not mimic the heart structures (ventricle/atrial patterning, heart cavity, for instance) and required speci c instrumentation for their analysis.Innervated muscle models, where motor neurons connect to skeletal muscle, have been achieved using the one-pot differentiation strategy from the neuromesodermal progenitors 10 .This demonstrated the possibility to study complicated and anatomically distal functional coupling in organoids.However, not many organs develop their cell types and innervation from a common progenitor, such as the neuromesodermal progenitor, thus this strategy is not easily transferrable to other organoids.The assembloid technology, on the other hand, presents an ideal modular platform for individual organoid components, that is suitable for innervation of organoids 2,3 .
Here, we describe a strategy to address the need of a method to innervate any organ type organoid relatively easily with symNs.As an example, we created cardiac organoids that are innervated by symNs.We purposely used a simple, easy to reproduce, and relatively low-cost assembly method, that does not require bioengineering or special instrumentation, with the goal for many researchers to be able to reproduce this technique.We created the assembloids in a modular way, so that researchers can adapt them to their needs.For example, one could use the symNs to innervate other organoids, such as lung, kidney, or liver.Or one could replace the symNs and innervate the cardiac organoids with parasympathetic neurons (SSRN: https://dx.doi.org/10.2139/ssrn.4318816) or sensory neurons 62,63 .
Organoids or assembloids must ful l certain criteria to be useful 3,28,40  which also displayed functional coupling regulation of CMs through symNs, inducible by nicotine 49 .In 2022, we described that symNs derived from iPSCs from the genetic autonomic disorder Familial Dysautonomia (FD) were hyperactive and in 2D co-cultures increased hPSC-derived CM beating 29 .Adding the option of a 3D organoid model to this toolset for disease modeling will increase its power for discovery of disease mechanisms and drug discovery.Our hSCAs are cultured in 3D throughout their generation and maturation stages (Fig. 1).( 2) Self-organization from stem or progenitor cells.Our hSCAs are assembled by mixing day 14 symNblasts and day 7 cardiac progenitors (Fig. 1).We 29,30 and others 35,36 have previously shown that these progenitors will form fully differentiated and functional symNs or cardiomyocytes upon continued 2D culture.Furthermore, these symNs have been employed by us to modeled autonomic dysfunctions in Familial Dysautonomia, within the SARS-CoV-2 infection milieu, and under diabetic hyperglycemia conditions 29,64,65 .(3) Contain multiple cell types that mimic the native organ.We show here that the hSCAs contain multiple cardiac and symN lineage cell types.The human heart consists of multiple cardiac lineages in addition to CMs, such as endothelial cells, smooth muscle cells, and cardiac broblast 33,34 .Our hSCAs contained mature cardiac muscle bers, T-tubules, cardiac broblasts, epicardial and endocardial layers, as well as atrial and ventricular CMs (Fig. 2b-d and   h-i, and 5f). 4. Mimicking some structural and functional features of the native organ.hSCAs contained CM-innervating functional symNs, in which NE synthesis was detected (Fig. 3a-e).The functional coupling between symNs and CMs was observed by Ca 2+ imaging and could be manipulated by nicotine and optogenetic stimulation (Fig. 3f-g).
It is becoming clearer that tissue innervation is essential for proper development, maturation and even repair of most organ tissues [66][67][68][69][70][71]  and the unknown effect of symN signaling to other cell types that form the heart mass 22,23 .Additionally, it is believed that such regulation on developmental cardiac hypertrophy by symNs is mediated by NE and adrenergic signaling 72,73 .Indeed, in 2022, Kowalski et al. co-cultured mouse primary symNs with hPSCderived CMs and showed that with symN innervation, the mature cardiac gene expression and functional cardiac activity were improved 53 .However, they also found that treating hPSC-derived CMs alone with isoproterenol, a β-adrenergic receptor agonist, was not su cient to induce such maturity improvement without physical connection with symNs 53 , implying undiscovered mechanisms in the symN-heart axis, which may also account for the reason of different heart sizes observed in the models above (P0 symN depletion in Kreipke et al., in which embryonic innervation remained, versus symN null in the heart in Tampakakis et al.).The hSCAs described here, therefore, might be an ideal model to assess development and reciprocal maturation of symNs and CMs in cultures.Using hSCAs, we modeled early heart development.Since the exact effect of NE on CMs is not fully clari ed 22,23,53 , we used α-and β-adrenergic receptor antagonist LAB to fully block the entire downstream target of NE (Fig. 4b).In our model, hSCA size and cell number were not altered in LAB-treated organoids compared to control (Fig. 4c).Additionally, genes for cardiac maturation decreased upon LAB treatment, indicating the importance of functional symN signaling for heart development and maturation.Future studies using hSCAs may focus on identifying the effect of other symN cofactors, such as neuropeptide Y on cardiac maturation, as well as the levels of cardiac maturation in different cardiac compartments using single cell RNA sequencing.
Finally, organoid technology has become an important tool for disease modeling approaches 4- 8,31,32,44,74 .In line with such studies, we employed our hSCAs to model the hypoxia-induced cardiac infraction (Fig. 5a).We successfully recapitulated the endogenous NE crisis in hSCAs from symNs upon hypoxic stress, which caused cardiac brosis that was rescued by treatment with the β blocker propranolol along with the hypoxic stress (Fig. 5b-g).There are a few limitations in this model.First, in the whole organism, other NE releasing tissues, such as adrenal chroma n cells, can also contribute to the NE crisis.Second, as a critical part during heart failure, the in ammation response was not recapitulated in hSCAs, due to the lack of immune cells in the organoids.Such challenges may be addressed in the future by incorporating more distal tissues, such as chroma n or immune cells into a further advanced assembloid.
Phalloidin staining (for F-actin) Phalloidin-iFluor 488 Reagent (Abcam, ab176753, 1:1000) was used according to manufacturer's instructions.Cryo-sectioned hSCAs were permeabilized and blocked by 0.2% Triton X-100 and 3% goat or donkey serum in PBS for 60 minutes, then incubated with 1x Phalloidin solution in PBS with 1% BSA for 30 minutes.Sections were washed by PBS for at least 3 times.General immunohistochemistry staining can be performed after Phalloidin staining.

Norepinephrine live labeling
The NE tracer (NS510) was a kind gift by Timothy Glass's laboratory at University of Missouri.hSCAs were incubated with 1 µM NS510 in hSCA culture medium and at 37°C for 60 min.After PBS wash for at least twice, hSCAs were imaged using Lionheart FX Automated Microscope at 440 nm excitation and 520 nm emission.

NE ELISA
NE assay was performed according to manufacturer's instructions (EagleBio, NOU39-K01).hSCA lysates from 1 well of 24-well plate were collected in 200 µl PBS.To preserve NE, sample stabilizer included in the kit was added to each sample.Lysate solutions were spun at 300 × g for 5 min to remove debris.The samples were ready for NE detection or were stored at − 80°C for long-term storage (although not recommended).

Light sheet microscopy
The imaging of the hSCAs was conducted using a custom-built light-sheet microscope 75 .The microscope was out tted with a 16x/0.8NA water-immersion detection objective (Nikon N16XLWD-PF).For the excitation of GFP uorescence, a 488 nm laser was employed, operating at a power density of 1.86 W/cm².Similarly, a 561 nm laser was used to stimulate RFP uorescence, also at a power density of 1.86 W/cm².The light sheet's thickness at the beam waist was 6.      Figure 5 hSCAs model hypoxia-induced infarction.

Figures
Figures

Figure 1 Assembly
Figure 1

Figure 4 SymNs
Figure 4 23Accordingly, symN innervation of the heart plays an important role in development and reciprocal maturation of the tissues.In 2015, Kreipke et al. used the neurotoxin 6hydroxydopamine (6-OHDA) to induce symN lesions in neonatal mouse hearts and found that the proliferation of CMs was increased, indicating disrupted cell cycle withdrawal due to the lack of symN innervation22.In 2021, Tampakakis et al. demonstrated similar results using a smooth muscle-speci c NGF deprivation mouse model, which resulted in heart-speci c symN depletion in embryonic hearts, as well as increased CM proliferation23.Interestingly, while both studies showed increased proliferation of CMs due to the absence of symN innervation, the model byKreipke etal.showed decreased heart size after symN depletion, whereas Tampakakis et al. showed enlarged heart size.This might be due to the difference in CM density (unchanged in Kreipke et al. and increased in Tampakakis et al.) in each model were xed in Trump's EM xative: [4% paraformaldehyde, 1% glutaraldehyde in 0.1M Phosphate buffer, pH 7.25] and washed several times in 0.1M Phosphate buffer before post-xation in 1% osmium tetroxide in buffer for 1 hour.The organoid samples were washed several times in deionized water and then placed in a 0.5% aqueous uranyl acetate enbloc for 1 hour in the dark.After several more washes in deionized water, the organoid samples were dehydrated in an ethanol series [30%, 50%, 75%, 95%, 100%], cleared in two changes of acetone, and two changes of propylene oxide.The organoid samples were in ltrated with 2:1, 1:1 and 1:2 mixtures of propylene oxide and Mollenhauer's Epon-Araldite plastic mixture 76 two hours respectively; then two changes of 100% Epon-Araldite plastic for at two hours each before embedding the tissues in at embedding molds.The embedded samples were polymerized in a 70-80° C oven overnight 77 .1µm sections from the polymerized blocks were obtained using a Reichert Ultracut S ultramicrotome.Sections were placed on glass and stained with 1% Toluidine Blue O in 1% sodium borate.The stained sections were evaluated, and areas of interest were chosen before trimming the corresponding block face for thin sectioning.60-70nm sections were obtained and placed on 200-mesh copper Locator grids.One of the grids was post stained with 2% aqueous uranyl acetate and Reynolds lead citrate (Reynolds, 1963), while the remaining grids were left unstained.Grids were viewed with a JEOL JEM-1011 transmission electron microscope at varying magni cations using an accelerating voltage of 100 KeV.Images were acquired using an AMT XR80M Wide-Angle Multi-Discipline Mid-Mount CCD Digital Camera with a resolution of 3296 x 2460 pixels.regulating cardiomyocyte endowment.Sci Transl Med 11. 10.1126/scitranslmed.aaw6419.74.Huch, M., Knoblich, J.A., Lutolf, M.P., and Martinez-Arias, A. (2017).The hope and the hype of organoid research.Development 144, 938-941.10.1242/dev.150201.75.Liu, Y., Song, M., Liu, B., and Kner, P. (2023).Single objective light sheet microscopy with multi- hSCAs hSCAs were xed by 4% PFA for 24 hours before AFM measurement.Stiffness was measured by AFM (Agilent Technologies 5500 Scanning Probe Microscope) using Aluminum coated cantilever (ASPIRE CCSR-10, spring constant = 0.1N/m).Fixed hSCAs were placed on the surface with minimal liquid approach for