Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

Patch-clamp Recordings and Single Fiber Labeling from Spiral Ganglion Somata in Acutely Prepared Semi-intact Cochleae from Neonatal Rats.

Spiral ganglion neurons (SGN) are the primary neuronal pathway for transmitting sensory information from the inner ear to the brainstem. Recent studies have revealed significant biophysical and molecular diversity indicating that auditory neurons are comprised of sub-groups whose intrinsic properties contribute to their diverse functions. Previous approaches for studying the intrinsic biophysical properties of spiral ganglion neurons relied on patch-clamp and molecular analysis of cultured somata that were disconnected from their pre-synaptic hair cell partners. In the absence of the information provided by cell-to-cell connectivity, such studies could not associate biophysical diversity with functional sub-groups. Here we describe a protocol for preparing, recording, and labeling spiral ganglion neurons in a semi-intact ex-vivo preparation. In these preparations, the cell bodies of spiral ganglion neurons remain connected to their hair cell partners. The recordings are completed within 4 hours of euthanasia, alleviating concerns about whether long culture times and culture conditions change the intrinsic properties of neurons.

Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells.

Sound intensity is encoded by auditory neuron subgroups that differ in thresholds and spontaneous rates. Whether variations in neuronal biophysics contributes to this functional diversity is unknown. Because intensity thresholds correlate with synaptic position on sensory hair cells, we combined patch clamping with fiber labeling in semi-intact cochlear preparations in neonatal rats from both sexes. The biophysical properties of auditory neurons vary in a striking spatial gradient with synaptic position. Neurons with high thresholds to injected currents contact hair cells at synaptic positions where neurons with high thresholds to sound-intensity are found in vivo. Alignment between in vitro and in vivo thresholds suggests that biophysical variability contributes to intensity coding. Biophysical gradients were evident at all ages examined, indicating that cell diversity emerges in early post-natal development and persists even after continued maturation. This stability enabled a remarkably successful model for predicting synaptic position based solely on biophysical properties.

Generation of inner ear hair cells by direct lineage conversion of primary somatic cells.

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

Worldwide, hearing loss is the most common loss of sensation. Most cases of hearing loss are due to the death of specialized hair cells found deep inside the ear. These hair cells convert sounds into nerve impulses which can be understood by the brain. Hair cells naturally degrade as part of aging and can be damaged by other factors including loud noises, and otherwise therapeutic drugs, such as those used in chemotherapy for cancer. In humans and other mammals, once hair cells are lost they cannot be replaced. Hair cells have often been studied using mice, but the small number of hair cells in their ears, and their location deep inside the skull, makes it particularly difficult to study them in this way. Scientists are seeking ways to grow hair cells in the laboratory to make it easier to understand how they work and the factors that contribute to their damage and loss. Different cell types in the body are formed in response to specific combinations of biological signals. Currently, scientists do not have an efficient way to grow hair cells in the laboratory, because the correct signals needed to create them are not known. Menendez et al. have now identified four proteins which, when activated, convert fibroblasts, a common type of cell, into hair cells similar to those in the ear. These proteins are called Six1, Atoh1, Pou4f3 and Gfi1. Menendez et al. termed the resulting cells induced hair cells, or iHCs for short, and analyzed these cells to identify those characteristics that are similar to normal hair cells, as well as their differences. Importantly, the iHCs were found to be damaged by the same chemicals that specifically harm normal hair cells, suggesting they are useful test subjects. The ability to create hair cells in the laboratory using more easily available cells has many uses. These cells can help to understand the normal function of hair cells and how they become damaged. They can also be used to test new drugs to assess their success in preventing or reversing hearing loss. These findings may also lead to genetic solutions to curing hearing loss.

Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.

In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell.

Global Ca2+ signaling drives ribbon-independent synaptic transmission at rod bipolar cell synapses.

Ribbon-type presynaptic active zones are a hallmark of excitatory retinal synapses, and the ribbon organelle is thought to serve as the organizing point of the presynaptic active zone. Imaging of exocytosis from isolated retinal neurons, however, has revealed ectopic release (i.e., release away from ribbons) in significant quantities. Here, we demonstrate in an in vitro mouse retinal slice preparation that ribbon-independent release from rod bipolar cells activates postsynaptic AMPARs on AII amacrine cells. This form of release appears to draw on a unique, ribbon-independent, vesicle pool. Experimental, anatomical, and computational analyses indicate that it is elicited by a significant, global elevation of intraterminal [Ca(2+)] arising following local buffer saturation. Our observations support the conclusion that ribbon-independent release provides a read-out of the average behavior of all of the active zones in a rod bipolar cell's terminal.