Search

Cell reprogramming is considered a stochastic process, and it is not clear which cells are prone to be reprogrammed and whether a deterministic step exists. Here, asymmetric cell division (ACD) at the early stage of induced neuronal (iN) reprogramming is shown to play a deterministic role in generating elite cells for reprogramming. Within one day, fibroblasts underwent ACD, with one daughter cell being converted into an iN precursor and the other one remaining as a fibroblast. Inhibition of ACD significantly inhibited iN conversion. Moreover, the daughter cells showed asymmetric DNA segregation and histone marks during cytokinesis, and the cells inheriting newly replicated DNA strands during ACD became iN precursors. These results unravel a deterministic step at the early phase of cell reprogramming and demonstrate a novel role of ACD in cell phenotype change. This work also supports a novel hypothesis that daughter cells with newly replicated DNA strands are elite cells for reprogramming, which remains to be tested in various reprogramming processes.

Epilepsy is a common chronic neurological disease affecting almost 3 million people in the United States and 50 million people worldwide. Despite availability of more than two dozen FDA-approved anti-epileptic drugs (AEDs), one-third of patients fail to receive adequate seizure control. Specifically, pediatric genetic epilepsies are often the most severe, debilitating and pharmaco-resistant forms of epilepsy. Epileptic syndromes share a common symptom of unprovoked seizures. While some epilepsies/forms of epilepsy are the result of acquired insults such as head trauma, febrile seizure, or viral infection, others have a genetic basis. The discovery of epilepsy associated genes suggests varied underlying pathologies and opens the door for development of new "personalized" treatment options for each genetic epilepsy. Among these, Dravet syndrome (DS) has received substantial attention for both the pre-clinical and early clinical development of novel therapeutics. Despite these advances, there is no FDA-approved treatment for DS. Over 80% of patients diagnosed with DS carry a de novo mutation within the voltage-gated sodium channel gene SCN1A and these patients suffer with drug resistant and life-threatening seizures. Here we will review the preclinical animal models for DS featuring inactivation of SCN1A (including zebrafish and mice) with an emphasis on seizure phenotypes and behavioral comorbidities. Because many drugs fail somewhere between initial preclinical discovery and clinical trials, it is equally important that we understand how these models respond to known AEDs. As such, we will also review the available literature and recent drug screening efforts using these models with a focus on assay protocols and predictive pharmacological profiles. Validation of these preclinical models is a critical step in our efforts to efficiently discover new therapies for these patients. The behavioral and electrophysiological drug screening assays in zebrafish will be discussed in detail including specific examples from our laboratory using a zebrafish scn1 mutant and a summary of the nearly 3000 drugs screened to date. As the discovery and development phase rapidly moves from the lab-to-the-clinic for DS, it is hoped that this preclinical strategy offers a platform for how to approach any genetic epilepsy.

Dravet syndrome is a catastrophic childhood epilepsy with early-onset seizures, delayed language and motor development, sleep disturbances, anxiety-like behaviour, severe cognitive deficit and an increased risk of fatality. It is primarily caused by de novo mutations of the SCN1A gene encoding a neuronal voltage-activated sodium channel. Zebrafish with a mutation in the SCN1A homologue recapitulate spontaneous seizure activity and mimic the convulsive behavioural movements observed in Dravet syndrome. Here, we show that phenotypic screening of drug libraries in zebrafish scn1 mutants rapidly and successfully identifies new therapeutics. We demonstrate that clemizole binds to serotonin receptors and its antiepileptic activity can be mimicked by drugs acting on serotonin signalling pathways e.g. trazodone and lorcaserin. Coincident with these zebrafish findings, we treated five medically intractable Dravet syndrome patients with a clinically-approved serotonin receptor agonist (lorcaserin, Belviq®) and observed some promising results in terms of reductions in seizure frequency and/or severity. Our findings demonstrate a rapid path from preclinical discovery in zebrafish, through target identification, to potential clinical treatments for Dravet syndrome.

Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms.

Zebrafish are a popular vertebrate model for human neurological disorders and drug discovery. Although fecundity, breeding convenience, genetic homology and optical transparency have been key advantages, laborious and invasive procedures are required for electrophysiological studies. Using an electrode-integrated microfluidic system, here we demonstrate a novel multichannel electrophysiology unit to record multiple zebrafish. This platform allows spontaneous alignment of zebrafish and maintains, over days, close contact between head and multiple surface electrodes, enabling non-invasive long-term electroencephalographic recording. First, we demonstrate that electrographic seizure events, induced by pentylenetetrazole, can be reliably distinguished from eye or tail movement artifacts, and quantifiably identified with our unique algorithm. Second, we show long-term monitoring during epileptogenic progression in a scn1lab mutant recapitulating human Dravet syndrome. Third, we provide an example of cross-over pharmacology antiepileptic drug testing. Such promising features of this integrated microfluidic platform will greatly facilitate high-throughput drug screening and electrophysiological characterization of epileptic zebrafish.

Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid diagnostics that can be administered at the point of care. Nucleic acid biomarkers for these diseases can be amplified and quantified via polymerase chain reaction (PCR). In order to solve the problems of conventional PCR--speed, uniform heating and cooling, and massive metal heating blocks--an innovative optofluidic cavity PCR method using light-emitting diodes (LEDs) is accomplished. Using this device, 30 thermal cycles between 94 °C and 68 °C can be accomplished in 4 min for 1.3 μL (10 min for 10 μL). Simulation results show that temperature differences across the 750 μm thick cavity are less than 2 °C and 0.2 °C, respectively, at 94 °C and 68 °C. Nucleic acid concentrations as low as 10(-8) ng μL(-1) (2 DNA copies per μL) can be amplified with 40 PCR thermal cycles. This simple, ultrafast, precise, robust, and low-cost optofluidic cavity PCR is favorable for advanced molecular diagnostics and precision medicine. It is especially important for the development of lightweight, point-of-care devices for use in both developing and developed countries.

Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC₅₀/EC₅₀) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.

Drug discovery and development to date has relied on animal models, which are useful but are often expensive, slow, and fail to mimic human physiology. The discovery of human induced pluripotent stem (iPS) cells has led to the emergence of a new paradigm of drug screening using human and disease-specific organ-like cultures in a dish. Although classical static culture systems are useful for initial screening and toxicity testing, they lack the organization of differentiated iPS cells into microphysiological, organ-like structures deemed necessary for high-content analysis of candidate drugs. One promising approach to produce these organ-like structures is the use of advanced microfluidic systems, which can simulate tissue structure and function at a micron level, and can provide high-throughput testing of different compounds for therapeutic and diagnostic applications. Here, we provide a brief outline on the different approaches, which have been used to engineer in vitro tissue constructs of iPS cell-based myocardium and liver functions on chip. Combining these techniques with iPS cell biology has the potential of reducing the dependence on animal studies for drug toxicity and efficacy screening.