Team:MIT mammalian

Implementing novel artifical sensors and actuators in the lab provides special challenges as all parts must be synthesized from scratch. Entire orthogonal pathways must be designed and made, and much consideration must be given to cross reactivity with different existing pathways that may lead to undesired effects in the synthetic circuit.

Mammalian cells, however, differentiate into materials of great complexity, sensitivity, and durability on a regular basis—-like muscle, bone, nervous tissue, and skin. All these actuators that take so much effort to create from scratch in the lab have been provided by nature in the form of genetic circuits and pathways contained in these cells. If we interface with mammalian cells at the right level and context with a toolkit of synthetic parts, we can selectively switch these pathways on or off. By taking advantage of the cell's built in sensors and actuators, we can direct differentiation into useful tissues, even 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!

In our project, we decided to link mechanical stimuli to osteogenesis. 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. There are a ton of applications for this technology; at the most basic level, one could imagine drawing a pattern onto a cellular monolayer and watch bone form around the outline. The system provides a very useful platform study morphogenesis and different types of mechanical signal transduction, a process little understood to date. 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 to the implementation of semi-orthogonal differentiation pathways and artifical organogenesis. We've developed a basic standard for linking mechanical sensing to cellular differentiation, a standard we hope to see it developed to support even more intricate systems.

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 control not only the proliferation and phenotype of a single cell, but also its interactions in a network of cells and eventually a network of different tissues. We need to control the spatial organization of these cells with respect to each other. This means we not only need to enable the cell to sense its surroundings and change its own phenotype accordingly, but also sense the phenotypes of the cells around it and act accordingly, whether it’s proliferation, differentiation, signaling, or apoptosis.

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.

LOOKING AHEAD

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.