July 1, 2014 (Vol. 34, No. 13)
Angela L. Huang, Ph.D.
Over the past decade, genetically encoded biosensors have proved to be powerful reporters of cellular activities, as shown using a variety of model organisms. They are highly amenable to customized designs to suit experimental needs, as they vary in fluorescence range, kinetics, and cellular compartmental targeting. They can be incorporated into a variety of cell types to support research into multiple physiological systems and processes. Although there are many immortalized human cell lines available, tissue-specific cell types that are not immortalized in vitro offer many experimental advantages, including retention of innate cellular functionalities that more accurately reflect in vivo human cellular functions.
At Tempo Bioscience, we developed a set of calcium and voltage biosensors that target different cellular compartments. We have incorporated these biosensors into human induced pluripotent stem cell (iPSC-) derived cell types, such as neural stem cells, astrocytes, and other neural cell types. Here, we report a new set of biosensor assays and show how they can be used for basic research, drug discovery, and chemical compound screening.
Currently, chemical dyes/sensors demonstrate difficulties targeting to specific cellular organelles and tend to compartmentalize randomly, before getting extruded from the cell during lengthy recording experiments (e.g., accumulation in cellular organelles). Genetically based biosensors can be tunable and more precisely targeted, using rational molecular and structural designs. The intensity changes at Excitation/Emission (Ex/Em) wavelengths are greater in magnitude for single-fluorophore reporters than traditional FRET-based biosensors. Unlike chemical biosensors, such as Fura-2, there is no ratiometric response, thereby making single-fluorophore biosensors more sensitive as reporters. This is advantageous for calibration and quantitative imaging measurements.
Assay Using TempoCal™, a Cytosolic Calcium Biosensor
Genetically encoded calcium sensors have become indispensable tools for cell biology, ligand-receptor studies, ion channel research, and drug discovery, but they suffer from the lack in varied biophysical and kinetic properties (e.g., wavelengths). TempoCal offers improved kinetics, with a Stoke’s Shift value of 15 nm, a single wavelength (610 nm Ex/ 625 nm Em; seconds-to-minutes response time) and intensiometric fluorescence indications. In Figure 1A and 1D, TempoCal-incorporated cells respond to 20 µM histamine, 20 µM carbachol, 10 µM ionomycin, and 2.5 mM CaCl2 with strong signal-to-noise cellular responses.
Assay Using TempoMito™, a Mitochondrial Calcium Biosensor
Mitochondria are responsible for cellular metabolism, toxicity, and signaling. The human central nervous system (CNS) is frequently affected in mitochondrial disorders (MCDs). CNS involvement in MCDs is manifested in conditions including epilepsy, stroke-like episodes, migraine, ataxia spasticity, psychiatric abnormalities, and neuropsychological deficits, just to name a few. Mitochondrial toxicity can be a side effect of drugs such as antivirals, antidepressants, and seizure medications. Commonly, cytotoxicity is manifested by calcium release from the mitochondria. We have engineered a calcium biosensor,
TempoMito, which targets to the mitochondria as a reporter for mitochondrial functionalities. Compared to chemical indicators, TempoMito can be expressed in a wide variety of human cell types. Figure 1B and 1D show that TempoMito-incorporated cells respond to standard activators such as histamine (20 µM) and calcium chloride (2.5 mM), as well as mitochondrial specific inhibitors (e.g., 20 µM carboxyamidotriazole, a known blocker for intracellular and mitochondrial calcium flux) at wavelengths of 610 nm/625 nm (Ex/Em).
Assay Using TempoVol™, a Voltage Cationic Biosensor
Cellular voltage biosensors are needed for a wide variety of cellular neuroscience studies in research and drug development, especially for ligand-gated ion channels, sodium, potassium, calcium, and chloride channels or transporters. They are tools for dissecting molecular mechanisms, as well as for observing cellular effects of chemical compounds. TempoVol is a reporter of ionic flux. Figures 1C and 1D show that TempoVol is responsive to pH changes, cellular calcium fluctuations, and the standard cation chelator, EDTA.
Why Use iPS-derived Human Cell Models with TempoCal, TempoMito, and TempoVol?
For decades, scientists have relied on immortalized human cell lines, biopsied from patients with various tumors, and the cells were immortalized using in vitro methods. Though primary human cells extracted from human tissues are ideal for research, rarely can researchers collect a sufficient amount of tissue of interest from patients or unaffected human subjects. Primary cell types can suffer from inconsistencies in vitro, which leads to various challenges in analyzing in vitro data. From the perspective of human genetics, diversity of human genetic backgrounds is an important factor in the evaluation of effects of chemical compounds, as is most eloquently illustrated by companion diagnostic tests for clinical drugs.
TempoCal, TempoVol, and TempoMito are biosensors incorporated into specific cell types of interest for basic research, preclinical drug discovery, and cellular toxicity screenings. They are functional reporters for the activation of target genes or pathways; they are also useful for screening for general cellular phenotypes such as altered intracellular calcium or pH and mitochondrial calcium toxicity.
At Tempo Bioscience, we believe that biosensors technologies embedded in human iPSC-derived cell types provide a new set of tools for scientists. They pave the way for developing standard screening platforms, where cells from genetically diverse human populations, using simple dermal-biopsy-based methods, can be further derived into useful tools. Our goal is to develop many cell types, with a CNS focus initially, and target biosensors into the cells to expand the number of drug discovery screening tools available to scientists.