Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental, Functional, and Terminal Toxicities in Neural Cells.

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells.

Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids.

Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics.

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids.

Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts.

Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures.

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture.

Mechanotransduction in cardiac hypertrophy and failure.

Cardiac muscle cells have an intrinsic ability to sense and respond to mechanical load through a process known as mechanotransduction. In the heart, this process involves the conversion of mechanical stimuli into biochemical events that induce changes in myocardial structure and function. Mechanotransduction and its downstream effects function initially as adaptive responses that serve as compensatory mechanisms during adaptation to the initial load.

Patient-Specific Induced Pluripotent Stem Cell Models: Generation and Characterization of Cardiac Cells.

The generation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes has been of utmost interest for the study of cardiac development, cardiac disease modeling, and evaluation of cardiotoxic effects of novel candidate drugs. Several protocols have been developed to guide human stem cells toward the cardiogenic path. Pioneering work used serum to promote cardiogenesis; however, low cardiogenic throughputs, lack of chemical definition, and batch-to-batch variability of serum lots constituted a considerable impediment to the implementation of those protocols to large-scale cell biology.

Modeling heart disease in a dish: from somatic cells to disease-relevant cardiomyocytes.

A scientific milestone that has tremendously impacted the cardiac research field has been the discovery and establishment of human-induced pluripotent stem cells (hiPSC). Key to this discovery has been uncovering a viable path in generating human patient and disease-specific cardiac cells to dynamically model and study human cardiac diseases in an in vitro setting. Recent studies have demonstrated that hiPSC-derived cardiomyocytes can be used to model and recapitulate various known disease features in hearts of patient donors harboring genetic-based cardiac diseases.

Moving to the core: spatiotemporal analysis of Forkhead box O (FOXO) and nuclear factor-κB (NF-κB) nuclear translocation.

Nuclear translocation of proteins is an essential aspect of normal cell function, and defects in this process have been detected in many disease-associated conditions. The detection and quantification of nuclear translocation was significantly boosted by the association of robotized microscopy with automated image analysis, a technology designated as high-content screening. Image-based high-content screening and analysis provides the means to systematically observe cellular translocation events in time and space in response to chemical or genetic perturbation at large scale.

High content screening: seeing is believing.

High content screening (HCS) combines the efficiency of high-throughput techniques with the ability of cellular imaging to collect quantitative data from complex biological systems. HCS technology is integrated into all aspects of contemporary drug discovery, including primary compound screening, post-primary screening capable of supporting structure-activity relationships, and early evaluation of ADME (absorption, distribution, metabolism and excretion)/toxicity properties and complex multivariate drug profiling. Recently, high content approaches have been used extensively to interrogate stem cell biology.

Understanding FOXO, new views on old transcription factors.

FOXO proteins are evolutionarily conserved transcription factors implicated in several fundamental cellular processes, functioning as end-point for transcriptional programs involved in apoptosis, stress response and longevity. Abrogation of FOXO function is very frequent in human cancer, therefore the mechanisms of regulation of the FOXO proteins are receiving increasing attention in cancer research. The FOXO proteins integrate regulatory inputs from a variety of upstream signaling pathways, most importantly in response to growth factor and stress signalling.

Adding more content to screening: reactivation of FOXO as a therapeutic strategy.

The discovery of novel targets that can be pharmacologically exploited to lead to a better disease outcome has long been an aim of biomedical research. At present, the technology and robotisation available have pushed the search for novel molecules to a high-throughput screening (HTS) context, making it possible to screen several hundreds of compounds or genes in a single day. High-content screenings (HCS) have added a refined complexity to the screening processes, as the information drawn from an image- based assay is more complete than the monoparametric readouts obtained in classical HTS assays.

Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.

Background: Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.

Results: The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers.

Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening.

FOXO proteins are direct targets of PI3K/Akt signaling and they integrate the signals of several other transduction pathways at the transcriptional level. FOXO transcription factors are involved in normal cell homeostasis and neoplasia, and they are regulated by multiple post-transcriptional modifications. In cancer research, the regulation of the FOXO factors is receiving increasing attention as their activation has been linked to cell-cycle arrest and apoptosis.

A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling.

Background: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells.

Methodology/principal Findings: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway.

An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays.

Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease-relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappaB, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks.