Princeton:Project Summary
From 2006.igem.org
PROGRAMMED DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING ARTIFICIAL SIGNALING PATHWAYS
Our vision is to develop reliable techniques for programmed tissue generation in mammalian systems. Our iGEM2006 work encompasses artificial cell-cell signaling and signal processing, directed differentiation, pattern formation, modeling and precise cellular placement.
MAMMALIAN SYSTEMS
Senders and Receivers
In order to program mammalian cell-cell communication, we are designing a synthetic sender-receiver system based on the LuxI-LuxR pathway. This detection pathway is responsive to acyl homoserine lactone (AHL). Cells engineered to be senders synthesize and secrete AHL which rapidly diffuses across the cellular membrane. Receivers detect the presence of AHL and react, depending on the programmed response of the cells. In our multiplexed system, senders and receivers are separate populations of engineered cells. A multiplexer allows us to choose between two different cell fates using only one inducer. We can imagine needing to differentiate two different tissue types in close proximity to one another in vivo, for example muscle and nerve tissue.
In our quorum sensing systems, the engineered cells are both senders and receivers. Quorum sensing gives us an additional level of control over how many cells we have and when they will differentiate. The toggle provides us with bistability after a transient application of inducer. This is advantageous in a living system because only a pulse of inducer is necessary for cells to differentiate into a programmed fate. Our two-step differentiation system adds an extra level of complexity and control.
Multiplexed Cell Fate Determination
Embryonic Stem (ES) cells are pluripotent, i.e. they can differentiate into any tissue in the body. We have developed a 1:2 multiplexer which pushes the cells into one of two different cell fates depending on the presence or absence of AHL. We are using two known master cell fate regulators (CFRs): MyoD and Ngn1. The expression of MyoD is activated in the presence of AHL while Ngn1 is activated in the absence of AHL. The expression of MyoD pushes ES cells toward a muscle fate (myocyte formation) and expression of Ngn1 pushes ES cells toward a neuronal fate. We are incorporating cell deposition techniques in order to precisely place senders and create controlled pattern formation of different tissues. For this we use Laser Direct Write (LDW) of cells which can accurately deposit the cells in specified locations. We are also investigating the use of microfluidics to deliver inducing agents in controlled locations.
Quorum Sensing Directed Toggle Switch
