A state of the art multiparametric flow cytometry analysis system for multidisciplinary stem cell research

Stem cells are capable of essentially extensive growth whilst retaining the ability to differentiate into most of the cell types found in the adult but unless we can find ways to grow them in large numbers while directing them to differentiate into therapeutically useful cells much of this potential will not be realised. A major obstacle impacting the derivation of differentiated cell types from stem cells is that current in vitro differentiation strategies often only produced the desired cells in small numbers. This means they must be separated from the other cell types present and one of the best techniques we have for doing this is flow cytometry. This technology relies upon the binding of fluorescent labelled antibodies to the surfaces of our desired cells such that when they pass through the flow cytometer they can be separated from the others and retain their viability. If appropriate antibodies are available, up to 12 different colours can be analysed at any one time using the instrumentation detailed in this proposal and this offers us an unparalleled advantage in cell identification and separation. There are a number of active research groups at the Institute of Human Genetics investigating a range of differentiation protocols designed to produce useful cells from a range of stem cells and flow cytometry instrumentation is not only indispensable to this work but is also subject to very heavy demand. In view of this, a successful outcome of this proposal would greatly enhance our ability to develop useful techniques for stem cell differentiation that will ultimately benefit medical science in general.

Stem cells, both embryonic and adult offer great promise for the development of therapeutic applications but for both of these stem cell types, separation of specific cells will be required. In the case of embryonic stem cells there is a need for culture strategies that can direct their differentiation towards therapeutically useful cells but currently available techniques produce heterogeneous mixtures from which we need to separate the desired cells. Adult stem cells exist in heterogeneous microenvironments or niches and again are often present in only very small numbers underlining the requirement for advanced separation technologies. Fluorescence activated cell sorting (FACS) is the most versatile method for separating such small numbers, provided that specific antibodies are available for the surface proteins of a specific cell type. A great advantage of FACS is that multiple antibodies labelled with different fluorochromes may be used in a single cell sample thereby increasing the total data available from a single experiment. Such multiparametric flow cytometry is cost effective in terms of antisera consumption and permits more detailed identification and isolation of rare cells but this requires the development of additional fluorescent labelled antibodies against proteins specific to these cells. This can only be achieved by increasing our understanding of stem cell biology which is a major focus of stem cell research at the Institute of Human Genetics. Stem cells of all types are difficult to grow in vitro hence a better understanding of the signal transduction mechanisms which promote pluripotency in embryonic stem cells and contribute to interaction with the niche for adult stem cells are an essential part of this proposal. Multiparametric flow cytometry will be an important part of this investigation since may use it for parallel examination of multiple intracellular proteins with small numbers of cells.