A novel characterisation and separation technique for pluripotent human embryonic and hematopoeitic stem cells

Realising the promised benefits of stem cells and their derivatives in regenerative medical therapies or in high throughput screening platforms for drug development necessitates the development of tools to purify cells to homogeneity. Ideally, such tools should be non-invasive with a capacity for separation several orders of magnitude beyond current methods. Industrial systems for the purification of therapeutic proteins provide precedents for large scale bioprocessing. To date, the vast majority of these have focused on column chromatography to achieve desired levels of purity. However, the size, sensitivity and complexity of cells present particular challenges to the downstream engineer which are unlikely to be solved by any further evolution or modification of traditional column chromatographic techniques. Consequently, novel techniques are needed. This proposal intends to develop a novel, simple, scalable and commercially useful technique for the separation/purification of human stem cells irrespective of their tissue of origin. This will be achieved by using Atomic Force Microscopy to define the topography and electrical charge distribution on the surfaces of human embryonic and adult haematopoietic stem cells followed by the use of this information in computational models to design complimentary surfaces. Surface prototypes will then be used in small-scale experimental work with living cells to demonstrate their ability to reversibly bind and separate cells and their subsequent viability. By focusing on both charge and topography of stem cell-surfaces the project will produce an adsorption-based separation technology more specifically suited for cell purification than current chromatographic techniques. The use of both adult and embryonic stem cell populations will exemplify the utility of this technology to both current and future clinical and research applications.

This proposal intends to develop a scalable separation/purification technique for human stem cells capable of non-invasive and reversible processing of cells that will supercede current methods based on flow cytometry, centrifugation or magnetic separation. By using Atomic Force Microscopy with charged tips to generate charge maps the research will identify surface electrical charge differences associated with specific human stem cell populations, namely embryonic and adult haematopoietic (CD34+) stem cells. This information will be used to computationally model complimentary interactive substrates providing reversible affinity. Small-scale experimental work will be used to evaluate substrate prototypes for their capacity to enrich for stem cell populations without altering their viability or pluripotency, assessed using a range of standardised in vitro assays for cell molecular marker expression, and differentiation potential. By focusing on both charge and topography of surfaces the work will produce an adsorption-based separation technology more specifically suited for cell purification than current chromatographic techniques. This novel technology should be scalable to processing larger quantities of cells (10 E9-10) than current methods, thus making it a commercially useful solid-phase separation technique.