QB3 Postdoc Seminar

Seminar | October 20 | 4:30-5:30 p.m. | 177 Stanley Hall

 QB3 - California Institute for Quantitative Biosciences

Speaker 1: Alison Walker (Miller lab)

Voltage imaging: New insights on neuronal network function

Neurons communicate over long distances via electrical pulses called action potentials. It is often the precise timing of action potentials that encode the various and far-reaching functions of our nervous systems. Our goal is to understand how multiple neuronal cell types, with intrinsically different properties, interact within a circuit. To do this, we have generated, characterized and applied the PeT-based voltage-sensitive dye, BeRST 1, in dissociated neuronal cultures. The photostability, fast kinetics and sensitivity of this dye are highly advantageous for recording spontaneous action potential firing of multiple neurons, an essential requisite to precisely dissect neuronal networks. Using pharmacological agents to probe network function as a whole, we find emergent changes in neuronal firing rate, which would not have been revealed by studying neurons individually, for example, with traditional patch-clamp electrophysiology. We are now extending our studies to assess the connectivity of every neuron within a network. This work depends on the creation of micro-patterned mini-networks and algorithms that assess spike timing, both of which are currently undergoing characterization.

Speaker 2: Nathaniel Huebsch (Healy lab)

Metabolically-Driven Maturation of iPS-Cell Derived Cardiomyocytes in Microphysiological Systems

Human induced pluripotent stem cell derived cardiomyocytes (iPS-CM) are a promising in vitro test-bed for drug development and cell-replacement therapy. However, like most other cell types derived in vitro from pluripotent stem cells, iPS-CM are immature, exhibiting morphology, physiology and pharmacology more reminiscent of the fetal, rather than the adult heart. In particular, the immature electrophysiology of iPS-CM suggests improper regulation of ion channels. Since drug binding to these channels are one of the most common causes of off-target drug toxicity, the use of immature cells with improper ion channel function limits the diagnostic utility of these cardiomyocytes, and may also pose risks of arrhythmias if these cells are transplanted into infarcted adult hearts.

Tissue engineering approaches involving aligned, 3D culture enhance iPS-CM maturation, but 3D culture alone is not sufficient to induce electrophysiological maturation. We hypothesized that recapitulating post-natal switching of the heart’s primary ATP source from glycolysis to fatty acid oxidation could enhance maturation of iPS-CM cultured in miniaturized 3D Microphysiological Systems (MPS). To test this hypothesis we first derived and purified iPS-CM from pluripotent stem cells. We also, independently, derived a defined, isogenic stromal cell population. To form micro-scale heart tissue, a defined cocktail of cells was injected into the culture chamber of micro-fabricated heart-on-a-chip platforms (Mathur et al. Sci. Rep. 2015). Physiology and pharmacology were assessed using motion tracking software (beating physiology; Huebsch et al. Tissue Eng. C. 2015), and other optical methods.

To identify media that could enhance electrophysiologic maturation, we used the robust design experimental methods to study the combinatorial effects of varying albumin, glucose, oleate and palmitate. After culturing MPS or monolayers for 3-14 days in these media, physiology and pharmacology were assessed. Consistent with previous observations of immaturity in iPS-CM, the action potential duration (APD) of MPS is prolonged when the system is cultured in standard cardiac media (> 600 ms). In contrast, MPS cultured in optimized maturation media exhibit APD similar to the APD of adult human left ventricle. Interestingly, the same media did not alter the APD of iPS-CM within monolayers. Matured Cardiac MPS exhibited pharmacology more reminiscent of Human Adult Left Ventricle compared to either basal Cardiac MPS or iPS-CM monolayers treated with the same media. However, both baseline and Matured Cardiac MPS were superior to iPS-CM monolayers in terms of their ability to predict safety margins for drugs that exhibit false positive toxicity (Verapamil) or false negative toxicity (Alfusozin) in traditional screening assays

This work shows that biophysical cues provided by Heart-On-A-Chip platforms combine synergistically with soluble metabolic cues to drive electrophysiological maturation of immature, pluripotent stem cell derived cardiomyocytes. Analysis of the underlying transcriptional mechanisms will likely guide rational design of conditions to push iPS-CM further toward a more “adult-like” state for more predictive in vitro modeling.