|Send to printer »|
GEN News Highlights : Jul 18, 2011
Scientists Develop Aptamer Sensor for Monitoring Single-Cell Interactions in Real Time
Approach involves attaching target-specific signaling aptamers to cell surface membrane.!--h2>
Researchers have developed an aptamer-based nanosensor that can be attached to the surface of living cells to monitor the interaction of individual cells with each other and with their environment in real time, both in vitro and, if early results can be built upon, in vivo. The team used the cell membrane-bound sensors to quantitatively detect the interaction of mesenchymal stem cells with their target molecules.
They suggest the generic approach can easily be modified to detect a range of cellular interactions. The investigators, who came from Brigham and Women’s Hospital (BWH), Harvard Medical School, Harvard Stem Cell Institute, and MIT, say the technique could feasibly be used in vivo to track the fate of transplanted cells and potentially even to determine drug response at the cellular level for individual patients. Details appear in Nature Nanotechnology in a paper titled “Cell-surface sensors for real-time probing of cellular environments.”
“Probing what cells ‘see’ and how they respond in real time to surrounding signals (i.e. cytokines) is still a major challenge,” note senior author Jeffrey Karp, Ph.D., co-director of the Center for Regenerative Therapeutics at BWH, and colleagues. In contrast with current technologies, Dr. Karp’s team has developed a sensor technology that covalently attaches fluorescent aptamers to the cell membrane and generates a real-time signal when the target molecule contacts the cell surface.
The researchers claim this approach is highly adaptable because high-affinity constructs of the single-stranded oligonucleotides to target molecules can be generated using an in vitro selection process known as systematic evolution of ligands by exponential enrichment, or SELEX.
For the published studies the team focused on attaching an aptamer that recognizes platelet-derived growth factor (PDGF) onto the surface membrane of mesenchymal stem cells (MSCs). PDGF is a chemoattractant that recruits MSCs to inflamed tissue and tumors, and represents a key signaling molecule in the participation of MSCs in vascular regeneration and communication with activated endothelial cells or tumor cells.
The PDGF-recognizing aptamer sensor is based on a construct that had previously been described, which comprised an aptamer carrying two different dye molecules, one at each end. When the sensor binds to PDGF a conformational change brings the dyes in close proximity, generating an instantaneous fluorescent signal. Although the sensor is highly sensitive and can detect PDGF in the picomolar range, the original construct was designed to work in the solution phase.
Dr. Karp’s team thus had to engineer the basic sensor to incorporate a surface anchoring moiety. They also carried out further changes in its base pair sequence to reduce background noise when used in physiological conditions such as the presence of divalent metal ions.
The scientists used this technique to develop two types of sensor: a quench sensor (FAM/Dabcyl), and a FRET sensor (Cy3/Cy5). The aim was to tag the cells using the sensors and then image transplanted MSCs using intravital confocal microscopy (IVM) to study intracellular signaling and cellular microenvironments in real time and at single-cell resolution.
Importantly, the researchers say the method for attaching sensors to the cell membrane doesn’t involve the complex genetic, enzymatic, or metabolic engineering methods used previously for modifying the cell surface. Instead, Dr. Karp et al’s procedure consists of three steps, which involve treating cell surface amines with sulphonated biotinyl-N-hydroxy-succinimide (NHS–biotin), followed by streptavidin-biotin interactions.
As an initial test to see whether the cell-bound sensors actually worked, they sensor-tagged MSC cells in vitro and added PDGF close to the cells. Fluorescence imaging showed spatial variation of the signal intensity over the cell surface, which changed over time as more PDGF was transported to the cells by the surrounding flow of fluid. They then used a previously described microwell assay to show that the sensor MSCs could detect PDGF secreted by neighboring cells.
The researchers moved on to evaluate the feasibility of using IVM to monitor the engineered cells after transplantation, as a step toward using cell surface sensors for monitoring the cellular environment, cell function, and intercellular signaling in vivo. They applied IVM to check whether sensor-tagging MSCs affected the cells’ natural homing ability to bone marrow in mice. Using a fluorescence confocal and multiphoton intravital imaging system that tracks single cells in living animals, they confirmed that the migration of sensor-engineered and native MSCs was no different.
The FRET system is useful for in vivo studies as it provides a ratio of two distinct fluorescent dyes and so minimizes the nonspecific background signal. With this in mind the team designed an oligonucleotide FRET-probe, to see whether a change in FRET signal could be detected in live mice. The FRET probe carries Cy3 and Cy5 in close proximity, which initially projects a Cy5 signal when exciting Cy3.
To investigate whether the FRET probe was viable in bone marrow in vivo, they carried out acceptor photobleaching of the Cy5 moiety in situ using a laser, which resulted in a switch from a Cy5 (red) to Cy3 (green) signal.
“This experiment clearly suggests that it is feasible to use IVM to track transplanted sensor-engineered cells and monitor their signaling with the target molecules in their in vivo niche environment,” the authors state. “We have shown that MSCs engineered with an aptamer can be detected in mouse bone marrow by IVM 24 hours post-transplantation, and the communication between FRET dyes is retained, suggesting that nucleic-acid-sensor conjugated fluorophores are functional on the cell surface for at least one day under physiological conditions.”
They suggest further protection from neuclase degradation in vivo could be engineered into the sensors using protective groups such as PEG, phosphorothioates, and locked nucleic acids. One of the key benefits of the cell surface sensor technique is that it detects local target molecule concentrations at, or very close to, the cell surface (the sensor ‘sticks out’ about 8 nm from the cell surface when bound to PDGF), which may be very different to the bulk concentration.
“Our approach may therefore provide significant advantages over in vitro techniques (such as ELISA) that can only assess bulk concentrations of cytokines and may serve as a new tool to quantitatively measure signaling molecules or potentially drugs at the cell surface with high spatiotemporal resolution under physiological conditions,” they claim.
The researchers’ ultimate goal is to use the natural homing ability of specific cell types to deliver sensors to specific tissues such as bone marrow, lympho nodes, sites of inflammation, or tumors, as a means to monitor intercellular communication in vivo. “Our ongoing work focuses on further engineering our FRET sensor to have a high signal-to-noise ratio under physiological conditions to monitor PDGF levels produced in the MSC niche by activated endothelial cells, tumor cells, or in response to injury.”
© 2013 Genetic Engineering & Biotechnology News, All Rights Reserved