Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.

We successfully determine the ranges of dielectric permittivity, cytoplasm conductivity, and specific membrane capacitance of mouse hippocampal neuronal and glial cells using dielectrophoresis (DEP) crossover frequency (CF). This methodology is based on the simulation of CF directly from the governing equation of a dielectric model of mammalian cells, as well as the measurements of DEP CFs of mammalian cells in different suspension media with different conductivities, based on a simple experimental setup. Relationships between the properties of cells and DEP CF, as demonstrated by theoretical analysis, enable the simultaneous estimation of three properties by a straightforward fitting procedure based on experimentally measured CFs. We verify the effectiveness and accuracy of this approach for primary mouse hippocampal neurons and glial cells, whose dielectric properties, previously, have not been accurately determined. The estimated neuronal properties significantly narrow the value ranges available from the literature. Additionally, the estimated glial cell properties are a valuable addition to the scarce information currently available about this type of cell. This methodology is applicable to any type of cultured cell that can be subjected to both positive and negative dielectrophoresis.

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Neuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (μ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an “en passant” synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.

In this work, we describe the separation of embryonic mouse hippocampal neurons from glial cells using a positive dielectrophoresis (DEP) process. Here, we have implemented a cell trapping-favorable, cell suspension solution with low conductivity. It enables positive dielectrophoresis for hippocampal neurons (thereby attracting them to the electrodes), while resulting in negative dielectrophoresis for glial cells (repelling them from the electrodes). We have systematically performed a mathematical simulation and analysis to anticipate the DEP frequency at which hippocampal neurons and glial cells are separated. Simulated DEP crossover frequencies have been experimentally verified, and new, refined glial dielectric and physical properties are suggested that better reflect the experimental results obtained. DEP movements of neurons and glial cells in targeted separation media were experimentally analyzed, under the specified electric signal. Additionally, we have confirmed our modeling results by selectively trapping neurons over electrodes on a custom-made, multi-electrode array (MEA), resulting in active recruitment of neurons over the stimulation and recording sites. This technique is a valuable addition to the toolbox for creating more functional and versatile multi-electrode arrays.