Mesenchymal stem cells (MSC) in the human hair follicle have been shown to be multi-potent and take part in cutaneous wound healing by providing new fibroblasts. They can be found in the dermalpapilla (DP) and the connective tissue sheath (CTS) which surrounds the hair follicle. Since they are easily accessible and are abundant, their harvesting is highly desired in regenerative medicine. Their isolation and characterization is however hindered by the lack of known specific antigens for identifying them, apart from the expression of nest in protein which is an intermediate filament also found in neuronal stem cells. Focal plane array (FPA) – fourier transform infrared (FTIR) spectroscopy provides a label-free and non-destructive method for obtaining macromolecular information from biological samples. As molecular bonds absorb infrared at specific wavelengths, and the level of absorbance depends on the quantity of the specific bonding, measurements of infrared absorbance provides qualitative and quantitative results of bio-molecules present in the sample. In order to identify and to characterizethe MSC, we applied FPA-FTIR to map scan over the centre of the hair bulb, over the DP and the CTS below the DP. The scanned area is sub-divided into 8192 small sub-sections, with each section approximating the size of a DP cell (5–10 lm) in situ. An FTIR spectrum is recorded in each sub-section, forming a hyper-spectral data cube. This hyper-spectral data cube was analyzed using the chemometric technique unsupervised hierarchical clustering analysis. Clustering individual spectra based on their chemical similarities. From this, bio-molecular information in each region could be extrapolated and de-convoluted to identify phenotypical traits. Based on the current knowledge on mesenchymal stem cells from the literature and from our nest in immunostaining results, we postulate that the MSC in the DP should be centered in the DP, contain high level of fatty acids (lipid reserve for when proliferation is required) and high level of proteins (signaling molecules and transcription factors are required to maintain stem cell state) and may be identified by the presence of phosphates (PO2-) related to DNA methylation state. By performing hierarchical clustering analyses on the spectra using different criteria, producing spectral clusters, and finding the region of high fatty acids and proteins,we successfully singled out an area which is likely to be the MSC-containing region within the DP. Individual spectra can be analyzed further for stem cell related features. We aim to verify this method as a valid way to narrowing down an MSC-containing region by staining the FTIR map scanned sample using anti-nestin antibody.
|Number of pages
|Published - 2010