Creating living materials which can remodel in response to external stress is a fascinating and widely pursued problem. Mammalian cells provide a vast array of built-in sensors and actuators. Interfacing with mammalian cells at the right level and context can allow us to take advantage of these complex native components. As proof of concept for this framework, we aimed to provide a system for touch-directed osteogenesis, or a "bone-forming touchpad". We progressed significantly on three fronts: successfully interfacing with the cell's built-in mechanosensitive genetic circuits, integrating chemical and mechanical signals via a bistable toggle to convert transient stimuli into permanent changes in cell state, and artificially directing osteogenesis. Preliminary testing of our system showed promising results. With these validated parts, we’ve established a toolkit for building an array of new useful cell lines and implementing novel circuits in mammalian cells using our new mammalian standard: the MammoBlock system. Our project set the stage for efficient post-iGEM integration and further characterization of these parts.
WHY MAMMALIAN CELLS?
Implementing new artifical sensors and actuators in the lab provides a special challenge as all parts must be synthesized from scratch. Entire orthogonal pathways must be designed, and consideration given to cross reactivity with different existing pathways.
Mammalian cells, however, differentiate into extremely complex and adaptable materials on a regular basis—-like muscle, bone, nervous tissue, and skin. All these material 'outputs' that would be difficult to create from scratch in the lab have already been provided by nature. If we can interface with mammalian cells using a toolkit of synthetic parts, we can selectively switch these pathways on or off. By taking advantage of the pathways provided by nature, we can direct differentiation, possibly even into synthetic organs!
Interfacing with the cell at the transcriptional level allows us to take full advantage of the built-in sensing and actuating circuits of the mammalian cell. Different signals are sensed by promoters in our circuit, which then integrates these signals and computes an output.
OUR PROJECT: A BONE-FORMING TOUCHPAD!
Our project began with idea of a biological touchscreen. We envisioned a cellular 'iPad', a plate of cells that could sense applied pressure and differentiate in response. Our system allows us to explore the role of chemical and mechanical signaling in differentiation by trying to build analogous synthetic counterparts, the first step towards artifical organogenesis.
In our system, cells sense transient mechanical stimuli in the form of either fluid shear stress, stretching, or substrate deformation. The stimuli is sensed by our circuit, flips a bistable toggle, and osteogenesis takes place in the cells that were subjected to mechanical forces.
Other important contributions from our project:
Canonical osteogenic factors in our new mammalian standard, MammoBlocks for easy assembly and incorporation into synthetic circuits.
For the first time: mechanosensitive promoters in addition to chemical-sensing promoters in MammoBlocks.
THE IMPORTANCE OF MECHANOSENSITIVITY
The path from cell to organ is complicated, and involves both rich chemical and mechanical feedback. To control organogenesis, we will need to be able to control a single cell and its interactions in a network of cells and different tissues. So we need to create cells that can sense the environment and the phenotypes of adjacent cells and respond by proliferating, differentiating, signaling, or apoptosing.
Recent paradigm shifted the importance of chemical signals (such as growth factors) to mechanical signaling, such as fluid shear stress, stretch, and substrate hardness. How do cells with the same genome form complex spatial structures? The canonical Turing model based on reaction diffusion is believed to give rise to cheeta spots and zebra stripes, but recently research has shown that this is not sufficient for making 3D structures. In fact, rich mechanical feedback is crucial in early embryogenesis to lay down basic structure of our body plans.
We cannot form complicated structures without mechanosensitivity! However, the current mammalian parts commonly used in synthetic biology does not allow for mechanosensitive parts. Our toolkit, developed during this summer through constructing our bone-forming touchpad, provides, for the first time, mechanosensitive promoters in addition to chemical-sensing promoters.
Tissue engineering is an emerging field, a creation of 21st century biology. A lot of the groundwork has been laid for the development of artificial organs; we’ve seen lab-grown bladders, heart cells beating in unison in a petri dish, and even human ear mimetics. So far, these organs have been created by populating artificial scaffolds with appropriately differentiated cells; the process has been controlled by the injection of cells into the right places at the right times. Here, we present a new take on manufacturing organs in vitro; an approach that requires cells to sense external stimuli and remodel the growing tissue accordingly.
We chose to explore in vitro bone tissue formation; however, the methods we develop here can be applied generally to the development of any artificial organ. Our specific goal is to create cells that sense mechanical stress, subcellularly integrate this information, and make a decision to differentiate into bone-producing osteoblast cells based on the external signals. These cells will be capable of ‘intelligent’ tissue formation; that is, when seeded onto a scaffold, they will create a bone fragment which is more dense in regions of higher mechanical stress.
The applications for cells with this capacity – to create tissue optimized to support a specific mechanical environment – are widespread. There’s the immediately obvious advantage of creating specialized bone grafts; in a nation that spends an average of $20 billion a year on treatments of bone-related fractures and replacements, increasing the efficiency of the bone surgeries would have a wide-ranging impact. But there’s another, more intriguing, application; a model system like this would allow us to directly study tissue development and organogenesis. By mimicking in vitro the natural processes of tissue remodeling, we can start to understand the challenges inherent in cell-dependent tissue structuring, and perhaps get a deeper grasp on the in vivo pathways involved.