Reinnervate

Research following the differentiation of cultured stem cells is heavily dependent on the expression of genes and proteins. Such molecules are frequently used to report on the developmental status of the culture as the cells sequentially pass through various stages of differentiation. Expression data is therefore important since it provides a snap shot of the progress of cellular differentiation and enables researchers to judge the direction of development and the types of tissues produced. Biomarkers of particular cell types are therefore valuable tools in stem cell science. Protein markers have been developed into immunological reagents that are marketed worldwide for use by biotechnologists and thus have significant commercial value.

Nonetheless, there are very few biomarkers that show an expression pattern that is exclusive to one particular cell type. For example, Nestin, a class III intermediate filament protein, is commonly used as a marker of neural progenitor cells. Nestin expression, however, is not exclusive to neural cells and has also been detected in testis, gut and pancreas, and more recently as a common marker of multilineage progenitor cells. Accordingly, the use of such biomarkers can lead to difficulties in an accurate assessment of cell differentiation status. This is particularly relevant to pluripotent stem cells that have the ability to form complex heterogeneous cultures consisting of a range of alternative tissue types.

Biomarker profiling of cell differentation:
To address this issue, ReInnervate is using advanced proteomic technologies to identify new biomarkers of stem cells and their differentiating derivatives. So called ‘biomarker’ profiling is becoming a popular new approach to track the progression of certain pathological conditions. ReInnervate is developing a unique application for this technology by using advanced mass spectrometry to follow the differentiation of stem cells. We have demonstrated that this approach is both accurate and reproducible (Hayman et al. 2004, 2006). There are several advantages to biomarker profiling:

1. The technology provides a lead into the identification of new proteins that are expressed in particular cell types. This is potentially of value when analysing a heterogeneous population of cells to determine the range of tissue types present.
2. The detection of many protein markers simultaneously provides greater accuracy in sample identification rather than measuring the expression of a single protein.
3. This method of detection and sample identification can be achieved rapidly (approximately 2 hours) which is significantly faster than more traditional methods examining protein expression such as western blot analysis.
4. Only small quantities of material are required for the generation of a mass spectrum. Monitoring the behaviour of cells in this way is versatile and could also be applied to assessing the status of cell differentiation in samples drawn from mass cultures and bioprocessors, especially when it is not possible to readily view the morphology of such cells.

Although protein profiling itself does not identify the individual molecules expressed in the cell sample this is not necessarily a concern for the initial application of this technology. A mass spectrum of proteins expressed in a cell sample represents a snap shot of the proteome at that specific point in time. The pattern of expression is a very dynamic parameter which depends on many factors including the history and status of the cells at the time of collection. Given such biological variation, subtle differences between samples of the same cell type(s) is inevitable and quantitative proteomic approaches should be used to account for such variation. Mass spectra expression profiles illustrate the complexity of the proteome, however, the generation of a peak map distils the frequently expressed molecules from the noise of molecules that show a more variable expression pattern. As a consequence, this provides an accurate map of the primary molecules consistently expressed by a particular type of culture.

ReInnervate is using mass spectra to identify proteins that are expressed in a reproducible and consistent manner. Additional technology involving direct amino-acid sequencing (e.g. tandem MS) and protein finger print analysis is being performed to identify candidate molecules and data are cross checked against sequence databases. The identification of novel proteins expressed in specific tissue types will be exploited, leading to the production of novel immunological reagents.

Markers of neural cell types:
Specifically, the Company is focusing on the identification of biomarkers associated with neural lineages, building on its expertise in developmental neuroscience. It is widely recognised that neural stem cells (NSCs), neuroprecursor cells (NPCs), and their differentiated derivatives (neurons and glia) will be useful for the drug discovery process and offer the potential to treat neurological disease. There are, however, few biomarkers of such cell types. In brief, we propose to use our expertise in neurobiology and apply modern proteomic approaches to identify novel markers of NSCs, NPCs and their derivatives. Antibodies will be raised against proteins that show selective expression in these cell populations. The identification of such molecules will be exploited and those markers expressed on the surface of cells can be used to specifically isolate viable normal human neural stem cells from complex cell mixtures. The identification and possible isolation of normal viable human neural stem/progenitors cells using novel antibody reagents will be of significant value to third parties such as pharmaceutical and biotechnology companies and academic researchers.

Development of diagnostic reagents:
Biomarkers of neural cell types, particularly immature cells, are also likely to have applications in the identification and monitoring of solid tumours in the brain and this will be of direct relevance to the healthcare industry. The identification of novel markers associated with type(s) of neural tumours (see below) will lead to the development of diagnostic and prognostic tools that can be used routinely for the identification and monitoring of neural tumours. Less intrusive tests (e.g. blood or urine sampling) using available clinical testing laboratories will be cost effective compared to existing methods (e.g. MRI) of monitoring neural tumour progression and/or recurrence. We have established links with neural surgeons and pathologists that supply samples of patient material (with the appropriate consent and ethical approval) to enable screening of neural tumours and corresponding blood samples to identify potential diagnostic / prognostic indicators.

Progress:
Research by ReInnervate’s scientists continues in this area and builds on the earlier development of this technology as described above. The Company has recently brought in specialists in advanced proteomic technology and protein biochemistry, and has access to state-of-the-art technology dedicated to biomarker identification. We also have access to and routinely use various types of mammalian stem cell and have in place the procedures to induce their differentiation into neural cell types. The identification of novel biomarkers for stem cells and their neural derivatives is well under progress. The Company envisages the out-licensing of this technology and the direct sale and/or licensing of any such immunological reagents and assays to bio-reagent supply companies to generate revenue streams as appropriate.

Related Links

Recent News