Development of stem cell marker technology based on photothermal nano-spectroscopy

Stem cells are cells that have the unique potential to produce any kind of cell in the body. They offer the potential to regenerate tissues and organs; each tissue has its own type of stem cell. In the future, a large number of medical conditions may be treated using stem cells, by using the patient's own cells to re-grow diseased or damaged tissue in the laboratory and transplanting the new tissue into the patient. Unfortunately, the location of many stem cell populations in the body are still not known. This is because stem cells are difficult to identify. Traditionally cell types have been identified by using markers, molecules which are unique to a particular type of cell; but no marker has been found which is unique for stem cells. Clearly, the fact that there is no single identifying marker for stem cells is a major handicap for future development in stem cell science. One tissue in which the location of stem cells is well known is the cornea, the transparent window at the front of the eye. The cells of the outermost surface of the cornea, termed corneal epithelium cells, only live for a few days, after which they die and are washed away by the tears. The stem cells, located in a ring around the edge of the cornea called the limbus, provide a continuous supply of fresh replacement cells to replace these. We plan to use this tissue to find potential SC markers, which will allow us to find stem cells in other tissues. The method we feel offers most promise to allow us to find a marker system for stem cells is infrared spectroscopy. Infrared is a type of light not visible to the human eye that when shined on a sample causes the molecules within the sample to vibrate; with different molecules vibrating at different frequencies. By measuring the frequencies of all the vibrations it is possible to obtain a unique spectral fingerprint for each sample. Currently, infra-red spectroscopy is not sensitive enough to analyse individual cells, so we propose to combine this technique with a relatively new type of microscope called a scanning probe microscope. These microscopes use very sharp needle-like probes to measure surface properties. Already, we have used a prototype instrument to measure clumps of a few cells and obtain fingerprints that clearly show that there are differences between stem cells and other cells. We will develop this prototype further, so that individual cells, and ultimately sub-cellular components like the nucleus, can be analysed. Using our detailed knowledge of where the cells in the cornea are, we will then analyse stem cells and their daughter cells, then produce a unique spectral fingerprint or marker for not only the stem cells but their daughter cells. In addtion order to test our new marker technology on the stem cells in the cornea, we will develop a system that will allow us to keep the cornea alive outside the body. This is called organ culture and it allows us to do experiments on isolated corneas.

It seems unlikely that a single marker for stem cells (SCs) will ever be found; currently the most promising approaches employ several markers in combination that, although not unique to SCs, are differentially expressed in comparison with other cell types. Thus it is essential that any new technology should have the ability to look at all of the components of the SCs simultaneously and the sensitivity to detect any changes to them. This is a difficult requirement to meet but one technique, which has the potential to do this, is Fourier-transform infrared spectroscopy (FTIR). Biomolecules absorb the mid-IR via vibronic transitions that are localised right down to individual chemical bonds, yielding richly structured 'fingerprint' spectra. All molecules within a cell contribute to the IR spectrum; giving a unique biochemical fingerprint of the cell. This technique has been used to detect the changes occurring in cells associated with diseases such as Alzheimer's, osteoporosis and to discriminate between malignant and non-malignant cells in several different tissues . Recently a new technique developed at Lancaster termed near-field photothermal micro spectroscopy has shown that it is possible to distinguish between different stages of the cell cycle and discriminate between SC's, transiently amplified (TA) cells and terminally differentiated (TD) cells. However, present state of the art in this field is not sufficiently sensitive to yet meet the stringent requirements of a stem cell marker technology. We propose to build on existing technology to develop stem cell marker technology based on IR spectroscopy. Our technique will be called photothermal nano-spectroscopy (PNS) technology. It will be non-diffraction limited, allowing nanoscale resolution, have the ability to obtain 3-D data from the cells and be benchtop based. We use the corneal epithelial stem cells as our model system as the location of the SC TA and TD cell populations are very well characterised in cornea. To facilitate our investigation we will develop a novel in vitro corneal culture system, which will allow us to study the SCs in situ in their stem cell niche for up to one month. It will also allow us to carry out BrdU chase experiments and to compare different species including bovine rat, rabbit and human. We will analyse our data using multivariate and vector analysis. The development of the PNS system together with our in vitro culture system and data analysis system will allow us to identify unique IR spectral markers for corneal adult stem cells, classify these markers biochemically and spatially, define the spectra components which change during the differentiation of daughter cells, and determine if the spectra markers are consistent across different species.