Team:MIT mammalian

From 2010.igem.org

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Our summer iGEM goal was to build a cellular touchpad, a cell line capable of responding to mechanical stimulus by differentiating into bone tissue. We split the project up into three parallel modules: mechanical sensing, signal processing and bone differentiation. For the mechanosensing portion of the project, we searched the literature for potential mechanosensitive promoters, then cloned them into expression vectors containing EGFP. We used plate shaking and microfluidic devices to mechanically stimulate the cells and screen for shear stress-responsive candidates. With signal processing, our goal was to convert a short pulse of mechanical stimulation into a permanent 'switch' for differentiation. We designed, built and tested a synthetic gene circuit controlled positive feedback of the rtTA3 transcription factor. The circuit showed robust upregulation after the activation of an inducible promoter.
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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.  
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For bone differentiation, we decided to create a dual cellular system. BMP2 (Bone Morphogenetic Protein) is a fast and efficient inducer of osteoblastogenesis; we plan to construct the cellular circuit in HEK cells, which are easier to engineer, and have them inducibly secrete BMP2 to differentiate co-cultured stem cells. We also managed to induce bone formation in two different stem cell lines, using human recombinant BMP2, and detect it on a western blot of cellular supernatant. In summary, we've accomplished what we set out to do - test mechanosensitive promoters, build a cellular circuit in mammalian cells, and induce bone differentiation in stem cells.  
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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.  
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Along the way, we also managed to create a new assembly standard for mammalian cells. 'MammoBlock' is a recombination-based protocol (see our New Mammalian Standard page for more information), especially useful when dealing with long mammalian construct sequences. It's a robust and efficient cloning procedure, that allows for quick creation of high-quality entry vectors. Like our morphogenetic toolkit, it is meant to act as the groundwork for future expansion in the world of mammalian synthetic biology.  
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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.  
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Revision as of 21:14, 26 October 2010

cellular touchpad

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.

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