Optical selection of stem cells: application to human embryonic germ cells

Human embryonic germ cells (EGCs) are capable of turning into many different types of cell, including ones with the potential to treat disease. For this reason, it is important that these cells are widely available to the research community. However, the difficulty of acquiring robust pure lines of EGCs has proven a significant barrier. The major problems for this are an extremely small mixed starting sample from which EGCs are made and a difficulty of propagating the delicate EGCs in laboratory culture. To overcome these difficulties, we need to a method to acquire pure EGCs and their precurors. Current methods are unsuitable. Microelectronics has provided us with very small scale devices capable of performing rapid, massive parallel processing of electrical signals. Likewise, optoelectronics has provided us with small scale devices capable of performing massive parallel processing of optical signals. The silicon technology that has been developed to create these tiny microelectronic devices, has in recent years, been applied to the manipulation and control of very small quantities of fluids. The beauty of this technology is the potential for an integrated single microstructured microchip. In Southampton we have recently developed optical chips for the transport of polymer beads around micrometer sized circuits. Light in the circuits carries these beads and can be switched so that they can be transported in one direction or the other. The speed of these beads depends upon their composition and size. We propose to transport cells in much the same way, thus different cells will be selectively 'pushed' by the light along the different tracks. Light is ideal for this purpose, especially since the wavelength to be used is not absorbed by the cell and thus will not damage it. The different cells will be identified by how quickly they are transported along the tracks. So we plan to identify EGCs without the need for any labels of fluorescent molecules - a significant improvement in the ease with which these cells can be purified. Once we have a pure population of cells for our experiments, we can discover which genes are switched on and off as EGCs are made and what factors we need to add to our laboratory cultures to keep EGCs growing. These advances will allow us to deposit our cell lines in the UK Stem Cell Bank so that other researchers can access them and the research community as a whole can make faster progress towards using stem cells to treat human disease.

The proposed programme of research, focused at the Engineering / Stem Cell Biology interface aims to advance human embryonic germ cell (EGC) research by microfluidic optical waveguide approaches for cell sorting. The EGC, a type of human pluripotent stem cell, is derived from the primordial germ cell (PGC) and is capable of broad differentiation. In spite of encouraging progress, major challenges still remain in researching EGCs. In particular, little is known of what regulates the conversion of PGC to EGC or, once formed, why EGCs struggle to self-renew in prolonged culture; both issues hinder their application to tissue engineering. Understanding derivation is inhibited by the tiny numbers of PGCs in the starting population that are mixed in with somatic cell types. These 'contaminating' somatic cells are carried over into EGC culture, making it difficult to ascribe precise reasons for the loss of self-renewal. A suitable method of delicate, non-invasive purification is needed as FACs and other conventional approaches are unsuitable because of the exceedingly limited cell number. We propose to develop optical sorting to purify PGCs and EGCs. Novel waveguides and microfluidic chambers will be designed and fabricated that are compatible with the sorting of living PGCs and EGCs. The result of this purification strategy, in addition to patentable technology applicable broadly across stem cell biology, will be to advance our EGC research. The acquisition of pure cells will allow: investigation of changes in gene expression that occur with the conversion of PGC to EGC; determination of the factors and signalling pathways that regulate EGC derivation and self-renewal; and investigation of whether pure populations of EGCs truly lack tumourigenicity in immunocompromised mice. Pure EGC lines will be generated, potentially with increased frequency. These will be deposited in the UK Stem Cell Bank where public access will facilitate expedited research on tissue engineering from EGCs towards therapeutic end-points.