Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Forward model for propagation-based x-ray phase contrast imaging in parallel- and cone-beam geometry.

We demonstrate a fast, flexible, and accurate paraxial wave propagation model to serve as a forward model for propagation-based X-ray phase contrast imaging (XPCI) in parallel-beam or cone-beam geometry. This model incorporates geometric cone-beam effects into the multi-slice beam propagation method. It enables rapid prototyping and is well suited to serve as a forward model for propagation-based X-ray phase contrast tomographic reconstructions. Furthermore, it is capable of modeling arbitrary objects, including those that are strongly or multi-scattering. Simulation studies were conducted to compare our model to other forward models in the X-ray regime, such as the Mie and full-wave Rytov solutions.

Mechanism of Permanganate-Promoted Dihydroxylation of Complex Diketopiperazines: Critical Roles of Counter-cation and Ion-Pairing.

The mechanism of permanganate-mediated dual C-H oxidation of complex diketopiperazines has been examined with density functional theory computations. The products of these oxidations are enabling intermediates in the synthesis of structurally diverse ETP natural products. We evaluated, for the first time, the impact of ion-pairing and aggregation states of the permanganate ion and counter-cations, such as bis(pyridine)-silver(I) (Ag+) and tetra- n-butylammonium (TBA+), on the C-H oxidation mechanism. The C-H abstraction occurs through an open shell singlet species, as noted previously, followed by O-rebound and a competing OH-rebound pathway. The second C-H oxidation proceeds with a second equivalent of oxidant with lower free energy barriers than the first C-H oxidation due to directing effects and the generation of a more reactive oxidant species after the first C-H oxidation. The success and efficiency of the second C-H oxidation are found to be critically dependent on the presence of an ion-paired oxidant. We used the developed mechanistic knowledge to rationalize an experimentally observed oxidation pattern for C3-indole-substituted diketopiperazine (+)-5 under optimal oxidation conditions: namely, the formation of diol (-)-6 as a single diastereomer and lack of the ketone products. We proposed two factors that may impede the ketone formation: (i) the conformational flexibility of the diketopiperazine ring, and (ii) hindrance of this site, making it less accessible to the ion-paired oxidant species.

Concise total synthesis of (+)-asperazine A and (+)-pestalazine B.

The highly convergent total synthesis of dimeric diketopiperazine alkaloids (+)-asperazine A and (+)-pestalazine B is described. A critical aspect of our expedient route was the development of a directed regio- and diastereoselective C3-N1' coupling of complex tetracyclic diketopiperazine components. This late-stage heterodimerization reaction was made possible by design of tetracyclic diketopiperazines that allow C3-carbocation coupling of the electrophilic component to the N1' locus of the nucleophilic fragment. The application of this new coupling reaction to the first total synthesis of (+)-asperazine A led to our revision of the sign and magnitude of the optical rotation for the reported structure.

Quantitative Modeling of Bis(pyridine)silver(I) Permanganate Oxidation of Hydantoin Derivatives: Guidelines for Predicting the Site of Oxidation in Complex Substrates.

The bis(pyridine)silver(I) permanganate promoted hydroxylation of diketopiperazines has served as a pivotal transformation in the synthesis of complex epipolythiodiketopiperazine alkaloids. This late-stage C-H oxidation chemistry is strategically critical to access N-acyl iminium ion intermediates necessary for nucleophilic thiolation of advanced diketopiperazines en route to potent epipolythiodiketopiperazine anticancer compounds. In this study, we develop an informative mathematical model using hydantoin derivatives as a training set of substrates by relating the relative rates of oxidation to various calculated molecular descriptors. The model prioritizes Hammett values and percent buried volume as key contributing factors in the hydantoin series while correctly predicting the experimentally observed oxidation sites in various complex diketopiperazine case studies. Thus, a method is presented by which to use simplified training molecules and resulting correlations to explain and predict reaction behavior for more complex substrates.

Pediatric evaluations for deep learning CT denoising.

BACKGROUND: Deep learning (DL) CT denoising models have the potential to improve image quality for lower radiation dose exams. These models are generally trained with large quantities of adult patient image data. However, CT, and increasingly DL denoising methods, are used in both adult and pediatric populations. Pediatric body habitus and size can differ significantly from adults and vary dramatically from newborns to adolescents. Ensuring that pediatric subgroups of different body sizes are not disadvantaged by DL methods requires evaluations capable of assessing performance in each subgroup. PURPOSE: To assess DL CT denoising in pediatric and adult-sized patients, we built a framework of computer simulated image quality (IQ) control phantoms and evaluation methodology. METHODS: The computer simulated IQ phantoms in the framework featured pediatric-sized versions of standard CatPhan 600 and MITA-LCD phantoms with a range of diameters matching the mean effective diameters of pediatric patients ranging from newborns to 18 years old. These phantoms were used in simulating CT images that were then inputs for a DL denoiser to evaluate performance in different sized patients. Adult CT test images were simulated using standard-sized phantoms scanned with adult scan protocols. Pediatric CT test images were simulated with pediatric-sized phantoms and adjusted pediatric protocols. The framework's evaluation methodology consisted of denoising both adult and pediatric test images then assessing changes in image quality, including noise, image sharpness, CT number accuracy, and low contrast detectability. To demonstrate the use of the framework, a REDCNN denoising model trained on adult patient images was evaluated. To validate that the DL model performance measured with the proposed pediatric IQ phantoms was representative of performance in more realistic patient anatomy, anthropomorphic pediatric XCAT phantoms of the same age range were also used to compare noise reduction performance. RESULTS: Using the proposed pediatric-sized IQ phantom framework, size differences between adult and pediatric-sized phantoms were observed to substantially influence the adult trained DL denoising model's performance. When applied to adult images, the DL model achieved a 60% reduction in noise standard deviation without substantial loss in sharpness in mid or high spatial frequencies. However, in smaller phantoms the denoising performance dropped due to different image noise textures resulting from the smaller field of view (FOV) between adult and pediatric protocols. In the validation study, noise reduction trends in the pediatric-sized IQ phantoms were found to be consistent with those found in anthropomorphic phantoms. CONCLUSION: We developed a framework of using pediatric-sized IQ phantoms for pediatric subgroup evaluation of DL denoising models. Using the framework, we found the performance of an adult trained DL denoiser did not generalize well in the smaller diameter phantoms corresponding to younger pediatric patient sizes. Our work suggests noise texture differences from FOV changes between adult and pediatric protocols can contribute to poor generalizability in DL denoising and that the proposed framework is an effective means to identify these performance disparities for a given model.

Multiple Kernel Synthesis of Head CT Using a Task-Based Loss Function.

In CT imaging of the head, multiple image series are routinely reconstructed with different kernels and slice thicknesses. Reviewing the redundant information is an inefficient process for radiologists. We address this issue with a convolutional neural network (CNN)-based technique, synthesiZed Improved Resolution and Concurrent nOise reductioN (ZIRCON), that creates a single, thin, low-noise series that combines the favorable features from smooth and sharp head kernels. ZIRCON uses a CNN model with an autoencoder U-Net architecture that accepts two input channels (smooth- and sharp-kernel CT images) and combines their salient features to produce a single CT image. Image quality requirements are built into a task-based loss function with a smooth and sharp loss terms specific to anatomical regions. The model is trained using supervised learning with paired routine-dose clinical non-contrast head CT images as training targets and simulated low-dose (25%) images as training inputs. One hundred unique de-identified clinical exams were used for training, ten for validation, and ten for testing. Visual comparisons and contrast measurements of ZIRCON revealed that thinner slices and the smooth-kernel loss function improved gray-white matter contrast. Combined with lower noise, this increased visibility of small soft-tissue features that would be otherwise impaired by partial volume averaging or noise. Line profile analysis showed that ZIRCON images largely retained sharpness compared to the sharp-kernel input images. ZIRCON combined desirable image quality properties of both smooth and sharp input kernels into a single, thin, low-noise series suitable for both brain and skull imaging.

A new method for the production of low-fat Cheddar cheese.

Our objective was to develop an alternative process to produce low-fat Cheddar cheese (LFCC) by combining reduced-fat Cheddar cheese (RFCC) made by a fat-removal process with micellar casein concentrate (MCC) to try to achieve the texture and flavor characteristics of full-fat Cheddar cheese (FFCC). The production of LFCC was replicated 3 times. The MCC was produced by ultrafiltration of skim milk, followed by 3 stages of microfiltration, and the final MCC was spray dried. The LFCC was formulated to achieve 6% fat, 28% protein, and 1.2% salt by a combination of RFCC, MCC powder, salt, and water. The 6% fat target was selected to comply with the FDA standard for a low-fat label claim. The pH of the LFCC mixture was adjusted to 5.3 by lactic acid. Rennet was added to the LFCC mixture, followed by pressing and packaging. Chemical and sensory data were analyzed by ANOVA using the Proc GLM of SAS to determine if any differences in chemical composition and sensory properties were present among different cheeses. Descriptive sensory scores were used to construct a principal component analysis biplot to visualize flavor profile differences among cheeses. The LFCC had 83% less fat, 32% less sodium, and higher protein and moisture content than FFCC. When the cheese texture was evaluated in the context of a filled-gel model consisting of matrix and filler (100% minus percentage of matrix) the LFCC had lower filler volume than FFCC, yet the LFCC had a softer texture than FFCC. The LFCC contained some of the original FFCC cheese matrix that had been disrupted by the fat-removal process, and this original FFCC matrix was embedded in the new LFCC matrix formed by the action of rennet on casein from the continuous phase of hydrated MCC. Thus, the texture of the LFCC was desirable and was softer than the FFCC it was made from, whereas commercial RFCC (50 and 75% fat reduction) were firmer than the FFCC. The sulfur flavor in LFCC was closer to FFCC than commercial RFCC. The LFCC had bitter and grape-tortilla off-flavors that came from the dried MCC ingredient. The commercial RFCC and LFCC made in this study were missing the typical aged Cheddar character (catty, nutty, fruity, brothy, milk fat flavors) found in aged FFCC. Future work to improve the flavor of LFCC made by the process described in this study should include the addition of a flavoring ingredient (e.g., enzyme-modified cheese) to enhance the aged Cheddar flavors and mask undesirable flavors.

An FDA Guide on Indications for Use and Device Reporting of Artificial Intelligence-Enabled Devices: Significance for Pediatric Use.

Radiology has been a pioneer in adopting artificial intelligence (AI)-enabled devices into the clinic. However, initial clinical experience has identified concerns of inconsistent device performance across different patient populations. Medical devices, including those using AI, are cleared by the FDA for their specific indications for use (IFUs). IFU describes the disease or condition the device will diagnose or treat, including a description of the intended patient population. Performance data evaluated during the premarket submission support the IFU and include the intended patient population. Understanding the IFUs of a given device is thus critical to ensuring that the device is used properly and performs as expected. When devices do not perform as expected or malfunction, medical device reporting is an important way to provide feedback about the device to the manufacturer, the FDA, and other users. This article describes the ways to retrieve the IFU and performance data information as well as the FDA medical device reporting systems for unexpected performance discrepancy. It is crucial that imaging professionals, including radiologists, know how to access and use these tools to improve the informed use of medical devices for patients of all ages.

Analysis of umbilical cord tissue as an indicator of in utero exposure to toxic adulterating substances.

In utero drug exposure is a significant public health threat to the well-being and normal development of the neonate. Recently, testing of umbilical cord tissue (UCT) has been employed to measure illicit drug exposure, as drugs used by the mother during the third trimester may be retained in the UCT. Focus has also been given to potential adverse health effects among drug users, resulting from exposure to pharmacologically active adulterants and cutting agents in the street drug supply. The in utero effects of these substances have not been well studied in humans, nor has their presence been demonstrated as a means for assessing adverse health effects in the neonate. Here, we describe the application of a novel test method to analyze UCT for the presence of more than 20 common adulterating/cutting substances via LC/Q-TOF. In total, 300 de-identified UCT samples were analyzed-all had previously tested positive for cocaine or opiates. Generally, the positivity rates of individual compounds were similar between the Cocaine and Opiates Subgroups, apart from levamisole, xylazine, dipyrone (metabolites), and promethazine. Many of the adulterants used in the street drug supply do have legitimate medicinal/therapeutic uses, including several of the compounds most frequently detected in this study. Caffeine and lidocaine were the most frequently identified compounds both individually (>70% each) and in combination with each other. Alternatively, levamisole, an adulterant with no legitimate therapeutic use, was present in 12% of cases. Importantly, this data demonstrates that the detection of traditional drugs of abuse may serve as indicators of potential in utero exposure to toxic adulterating substances during gestation. While there is cause for concern with respect to any unintentional drug exposure, illicit drug use during pregnancy, including uncontrolled dosing, poly-adulterant consumption, and the interactions of these drug mixtures, produces a significant public health threat to the neonate which warrants further study.

Qualitative LC-Q-TOF Analysis of Umbilical Cord Tissue via Data-Dependent Acquisition as an Indicator of In Utero Exposure to Toxic Adulterating Substances.

Toxic adulterants are drug or chemical agents used to add bulk volume to traditional drugs of abuse such as cocaine and heroin. These cutting agents include levamisole, metamizole, noxiptillin, phenacetin and xylazine as well as common legal drugs such as acetaminophen, caffeine, diphenhydramine, lidocaine, quinine, quetiapine and tramadol. Because they possess pharmacological activity they result in exposure of the user, but also in the case of pregnant women, the developing fetus, to potential drug toxicity. We describe the development, validation and implementation of a rapid (48 second sample-to-sample) test based on a qualitative data-dependent liquid chromatography-quadrupole time of flight mass spectrometry method for the analysis of toxic adulterating substances in umbilical cord tissue (UCT) samples. The method provides a means of studying potential in utero exposure to these agents. Library spectra comparison at three different collision energies was used in conjunction with retention time and accurate mass to identify these substances in UCT. Analytically based reporting limits were established to determine positivity rates of adulterants in UCT utilizing a standard addition approach. The method was applied to authentic cocaine and opioid positive UCT to screen for toxic adulterants. There were a total of 82 potential adulterant positives found in a 30-sample cohort of authentic UCT samples, with an average of 2.7 substances per case. Lidocaine was the predominant finding followed by caffeine, and diphenhydramine all of which could result from non-illicit drug exposure, however, there were positives for levamisole, phenacetin, noxiptillin and xylazine none of which are approved in the United States for human therapeutic use. This initial set of data established a preliminary positivity rate of potentially toxic adulterants in UCT samples positive for cocaine or opioid use.

2,2,2-Tris(pyrazol-1-yl)ethanol.

The title compound TPE, C(11)H(12)N(6)O, was prepared by slow evaporation from diethyl ether. In the crystal, there is a hydrogen bond between the alcohol H atom and an N in the pyrazole ring of a neighboring mol-ecule.

Empirical beam hardening and ring artifact correction for x-ray grating interferometry (EBHC-GI).

PURPOSE: Talbot-Lau grating interferometry enables the use of polychromatic x-ray sources, extending the range of potential applications amenable to phase contrast imaging. However, these sources introduce beam hardening effects not only from the samples but also from the gratings. As a result, grating inhomogeneities due to manufacturing imperfections can cause spectral nonuniformity artifacts when used with polychromatic sources. Consequently, the different energy dependencies of absorption, phase, and visibility contrasts impose challenges that so far have limited the achievable image quality. The purpose of this work was to develop and validate a correction strategy for grating-based x-ray imaging that accounts for beam hardening generated from both the imaged object and the gratings. METHODS: The proposed two-variable polynomial expansion strategy was inspired by work performed to address beam hardening from a primary modulator. To account for the multicontrast nature of grating interferometry, this approach was extended to each contrast to obtain three sets of correction coefficients, which were determined empirically from a calibration scan. The method's feasibility was demonstrated using a tabletop Talbot-Lau grating interferometer micro-computed tomography (CT) system using CT acquisitions of a water sample and a silicon sample, representing low and high atomic number materials. Spectral artifacts such as cupping and ring artifacts were quantified using mean squared error (MSE) from the beam-hardening-free target image and standard deviation within a reconstructed image of the sample. Finally, the model developed using the water sample was applied to a fixated murine lung sample to demonstrate robustness for similar materials. RESULTS: The water sample's absorption CT image was most impacted by spectral artifacts, but following correction to decrease ring artifacts, an 80% reduction in MSE and 57% reduction in standard deviation was observed. The silicon sample created severe artifacts in all contrasts, but following correction, MSE was reduced by 94% in absorption, 96% in phase, and 90% in visibility images. These improvements were due to the removal of ring artifacts for all contrasts and reduced cupping in absorption and phase images and reduced capping in visibility images. When the water calibration coefficients were applied to the lung sample, ring artifacts most prominent in the absorption contrast were eliminated. CONCLUSIONS: The described method, which was developed to remove artifacts in absorption, phase, and normalized visibility micro-CT images due to beam hardening in the system gratings and imaged object, reduced the MSE by up to 96%. The method depends on calibrations that can be performed on any system and does not require detailed knowledge of the x-ray spectrum, detector energy response, grating attenuation properties and imperfections, or the geometry and composition of the imaged object.

Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

Wave optics simulation of grating-based X-ray phase-contrast imaging using 4D Mouse Whole Body (MOBY) phantom.

PURPOSE: Demonstrate realistic simulation of grating-based x-ray phase-contrast imaging (GB-XPCI) using wave optics and the four-dimensional Mouse Whole Body (MOBY) phantom defined with non-uniform rational B-splines (NURBS). METHODS: We use a full-wave approach, which uses wave optics for x-ray wave propagation from the source to the detector. This forward imaging model can be directly applied to NURBS-defined numerical phantoms such as MOBY. We assign the material properties (attenuation coefficient and electron density) of each model part using the data for adult human tissues. The Poisson noise is added to the simulated images based on the calculated photon flux at each pixel. RESULTS: We simulate the intensity images of the MOBY phantom for eight different grating positions. From the simulated images, we calculate the absorption, differential phase, and normalized visibility contrast images. We also predict how the image quality is affected by different exposure times. CONCLUSIONS: GB-XPCI can be simulated with the full-wave approach and a realistic numerical phantom defined with NURBS.

Phase-contrast imaging with a compact x-ray light source: system design.

X-ray phase-contrast imaging (XPCI) overcomes the problem of low contrast between different soft tissues achieved in conventional x-ray imaging by introducing x-ray phase as an additional contrast mechanism. This work describes a compact x-ray light source (CXLS) and compares, via simulations, the high quality XPCI results that can be produced from this source to those produced using a microfocus x-ray source. The simulation framework is first validated using an image acquired with a microfocus-source, propagation-based XPCI (PB-XPCI) system. The phase contrast for a water sphere simulating a simple cyst submersed in muscle is evaluated and the evolution of PB-XPCI signal as the object to detector distance is increased is demonstrated. The proposed design of a PB-XPCI system using the CXLS is described and simulated images of a coronary artery compared between CXLS and microfocus source PB-XPCI systems. To generate images with similar noise levels, a microfocus source would require a 3000 times longer exposure than would the CXLS. We conclude that CXLS technology has the potential to provide high-quality XPCI in a medical environment using extremely short exposure times relative to microfocus source approaches.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells.

THREE MODES FOR CRYOPRESERVATION (CP) OF HUMAN IPSC CELLS HAVE BEEN COMPARED: STD: standard CP of small clumps with 10% of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze(®) concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.

Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.

Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.