Characterization of human corneal stem cells by synchrotron infrared micro-spectroscopy

Abstract

Purpose: The purpose of this study was to use high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy coupled with multivariate analysis to investigate the characteristics of adult stem cell (SC) and transit amplifying (TA) cell populations of the human corneal epithelium.

Methods: Spectra of individual SC and TA cells in situ from cryosections of human cornea were collected using a synchrotron micro-spectroscopy facility at Daresbury laboratory, UK. Multivariate analysis and Mann Whitney U tests were used to analyse the spectral data from the SC and TA cell populations.

Results: There were marked differences between the median spectra of the two cell populations. This correlated with their level of differentiation and functional specialization. Multivariate (principal component) analysis revealed that the cell populations could be segregated into distinct clusters, with only slight overlap between the two cell types. Significant (p<0.05) spectral differences were found in the spectral regions associated with nucleic acid, protein and lipids.

Conclusions: Synchrotron FTIR micro-spectroscopy together with principal component analysis is able to discriminate between SC and TA cell populations. Our results also suggest a small sub-population of corneal epithelial cells in the SC niche have TA cell-like characteristics. Many of the spectral differences between the SC and TA cell populations relate to differences in nucleic acid conformation.

Figure 1

Schematic diagram the corneal epithelium showing the location of the SC and TA cell populations. The stem cells (red) are the basal cell layer located at the limbus; these stem cells proliferate and produce transit amplifying cells (blue) which migrate away from the limbus onto the cornea.

Figure 2

Light micrographs of the corneal and limbal epithelium. A: Light micrograph of the corneal epithelium. The TA cells are the basal cell layer (asterisk) below which is Bowman's layer (arrow). B: Light micrograph of the limbal epithelium. The invaginations of the basal lamina in the limbal region are termed the palisades of Vogt (arrow). The stem cells are located in the basal cell layer and are concentrated at the base of the rete ridge-like structures (asterisk). The scale bars are equal to 100 μm.

Figure 3

Three dimensional cluster plot of the spectral PCs for SC and TA cell populations. This figure shows a three dimensional cluster plot of the spectral PCs for SCs (red) and TA cells (blue). Nearness of points implies pattern recognition and the separation of sample clusters in the plots signifies structurally dissimilar groups. It is evident that the two populations of cells form distinct clusters, although the discrimination is not perfect.

Figure 4

The SC and TA cell median spectra, the difference spectra, and the statistically significant regions of SC and TA cell spectra. Synchrotron FTIR microspectrosopic comparison of SC and TA cell populations in human cornea (A). Median spectra for SC (red) and TA (blue) cell populations. B: The difference spectra between the SC and TA cell populations. C: Significance values for Mann-Whitney comparison of SC and TA spectra (p<0.05 below line).