Enabling Technologies for Stem Cell Biology

We are interested in understanding what extrinsic factors control stem cell phenotype, specifically as it pertains to embryonic stem cell (ESC) self-renewal and differentiation. While biologists understand many aspects of self-renewal, especially in mouse ESCs, the effects of cell-secreted autocrine and paracrine factors are not well understood.

Cell patterning -- One technology that we have been developing allows us to place single ESCs in defined locations, giving us excellent control over cell microenvironment. This technology uses DEP traps to create potentially energy wells for cell patterning. We have used quantitative modeling to develop a simple yet suprisingly strong planar DEP trap for single-cell patterning.

We have also developed a non-electrical approach to cell patterning where the user simply flips a chip containing cells in wells onto another substrate, whereby the cells fall onto the second substrate and are patterned. The bio flip-chip allows patterning of cells without patterning of substrate, can pattern cells onto other cells or ECM patterns, and can pattern cells over topography. It is ideally suited for patterning embryonic stem cells, which proliferate rapidly into colonies to migrate over the substrate. Below is a pattern of mouse ESCs over two days, showing that colonies can grow and proliferate normally.

We are using cell patterning to study cell-cell signaling in mESC proliferation and self-renewal. Independently controlling local and global cell density provides a convenient platform for studying cell-cell signaling.

Microfluidic perfusion culture -- A second technology we have developed allows us to culture cells under flow in order to modulate diffusible signals. Our approach uses a microfluidic culture chambers where we can vary flowrate and/or reagent concentration to control the media around the cells and cell-cell soluble signaling. These devices include valves, debubblers, and other features to enable robust multi-day on-chip culture. Below we show an image of a recent device that incorporates two sets of three chambers to allow running of multiple conditions on one chip. At right is an image of mESCs growing in chambers of different flowrates.

We are using this system to study how diffusible signaling affects neuronal differentiation, and specifically the role of autocrine factors in neuronal differentiation.

Microfluidic cell pairing and fusion -- We have also been developing a device that allows us to pair and then fuse thousands of cells in parallel. The application is to understand fusion-mediated nuclear reprogramming of somatic cells, where somatic celss, upon fusion to embryonic stem cells, become pluripotent. Current techniques to perform such fusions have poor pairing and fusion efficiencies, leading to heterogeneous cultures. The device uses silicon capture combs (SEM at right) and a three-step loading procedure to pair thousands of cells in parallel. We can then use either PEG or electrofusion to fuse cells ,attaining extremely high fusion efficiencies (~80% for electrofusion). We are using this device to study the biology of fusion-mediated reprogramming, and it has other applications in immunology and in studying heterotypic cell interactions.