The design of synthetic developmental programs for multicellular mammalian systems is a cornerstone of synthetic biology. The programs hinge on the construction of gene regulatory circuits that control the patterning and morphogenesis of synthetic multicellular structures. However, how the nongenetic properties of the growth environment impact circuit behavior remains poorly understood. Now, in a new paper, researchers show that cell density modulates the transduction of signal between a sender and receiver cell. In both computational models and laboratory experiments, researchers used cell density as an effective tool for controlling how mouse cells pattern themselves into complex structures.
This work is published in Nature Communications in the paper, “Control of spatio-temporal patterning via cell density in a multicellular synthetic gene circuit.”
“This paper represents progress towards our big picture goal of engineering synthetic tissues,” said Leonardo Morsut, PhD, an assistant professor of stem cell biology and regenerative medicine, and biomedical engineering at the Keck School of Medicine of USC. “Synthetic tissues could have endless medical applications, ranging from testing potential drugs or therapies to providing grafts or transplants for patients.”
The study used two types of mouse cells—connective tissue cells and stem cells—engineered to carry a “synNotch” genetic circuit which is based on notch signaling. SynNotch circuits have emerged “as a modular and flexible strategy for engineering multicellular mammalian systems.” Located on a cell’s surface, this protein-based sensor recognizes an external signal that triggers the cell to respond—usually by turning on a user-defined gene.
For these experiments, the scientists used synNotch to turn on a circuit that includes green fluorescence and a way to propagate the signal further. The fluorescence made it easy to observe cells as they formed patterns. While conducting these experiments, Marco Santorelli, PhD, a postdoc in the Morsut Lab, noticed that genetically identical cells did not always produce the same patterns.
“We would see different outcomes of the patterning when we would start with genetically identical cells in different numbers,” said Morsut. “So that was puzzling at the beginning. I remember Marco came in and told me once that the experiment worked, but only in half of the plate. And when we looked at it more carefully, we started seeing that there was a gradient of cell density that seemed to correlate with differences in patterning.”
Above a certain cell density, synNotch exerted a weaker effect and didn’t produce the same patterns. Further complicating matters, cell density constantly shifted as cells proliferated at ever-changing rates—interacting in complex ways with the synNotch genetic circuit.
Pranav S. Bhamidipati, a candidate in the USC-Caltech MD-PhD program, built a computational model that could predict and clarify this complex and dynamic cell behavior.
“For me, this was one of the first times in my life where computational modeling has been able to predict behaviors that look like what actually happens in the cells,” said Matt Thomson, PhD, an assistant professor of computational biology at Caltech and an investigator with the Heritage Medical Research Institute. “Here, it helped guide us to think about how the cell density, proliferation rate, signaling, and all these different things conspire.”
“We were happy that we had the computational model to really explore and get a sense of what are the possible different patterns,” noted Morsut, “and how to move from one to another.” Guided by the computational model, the scientists were able to use cell density to generate a variety of predictable fluorescent patterns that developed over specific timeframes.
To understand how cell density was exerting these effects, a series of experiments yielded a surprising discovery. Greater cell density induces stress that leads to a quicker breakdown of not only synNotch, but also cell surface sensors in general.
Cell density is a broadly applicable tool for guiding both engineered and naturally occurring cells to build a vast array of structures, tissues, and organs. “Nature has relied on cell density in conjunction with genetic circuits to generate the remarkable diversity of multicellular structures, tissues, and organs,” said Morsut. “Now we can co-opt this same strategy to advance our efforts to build synthetic multicellular structures—and eventually tissues and organs—for regenerative medicine.”
