Microspectroscopy of spectral biomarkers associated with human corneal stem cells

Abstract

Purpose: Synchrotron-based radiation (SRS) Fourier-transform infrared (FTIR) microspectroscopy potentially provides novel biomarkers of the cell differentiation process. Because such imaging gives a "biochemical-cell fingerprint" through a cell-sized aperture, we set out to determine whether distinguishing chemical entities associated with putative stem cells (SCs), transit-amplifying (TA) cells, or terminally-differentiated (TD) cells could be identified in human corneal epithelium.

Methods: Desiccated cryosections (10 microm thick) of cornea on barium fluoride infrared transparent windows were interrogated using SRS FTIR microspectroscopy. Infrared analysis was performed through the acquisition of point spectra or image maps.

Results: Point spectra were subjected to principal component analysis (PCA) to identify distinguishing chemical entities. Spectral image maps to highlight SCs, TA cells, and TD cells of the cornea were then generated. Point spectrum analysis using PCA highlighted remarkable segregation between the three cell classes. Discriminating chemical entities were associated with several spectral differences over the DNA/RNA (1,425-900 cm(-1)) and protein/lipid (1,800-1480 cm(-1)) regions. Prominent biomarkers of SCs compared to TA cells and/or TD cells were 1,040 cm(-1), 1,080 cm(-1), 1,107 cm(-1), 1,225 cm(-1), 1,400 cm(-1), 1,525 cm(-1), 1,558 cm(-1), and 1,728 cm(-1). Chemical entities associated with DNA/RNA conformation (1,080 cm(-1) and 1,225 cm(-1)) were associated with SCs, whereas protein/lipid biochemicals (1,558 cm(-1) and 1,728 cm(-1)) most distinguished TA cells and TD cells.

Conclusions: SRS FTIR microspectroscopy can be employed to identify differential spectral biomarkers of SCs, TA cells, and/or TD cells in human cornea. This nondestructive imaging technology is a novel approach to characterizing SCs in situ.

Figure 1

Immunolabeling for the presence or absence of stem cells (SC) in human cornea. A: Section of limbal and peripheral cornea immunolabeled with a green fluorescent marker (Keratin 15) for corneal SCs. The cell nuclei have been stained with DAPI (4',6-diamidino-2-phenylindole; blue) and the basement membrane immunolabeled for laminin (red). B: Schematic diagram of the immunolabeled section with putative SCs labeled green, migrating right along the basement membrane to the transit-amplifying cell region and then upwards to surface to the terminally-differentiated cell region.

Figure 2

Ultrastructural analysis of cells types in human corneal epithelium. A: Transmission electron micrograph (TEM) of terminally-differentiated corneal epithelial cells. Cells on the surface were highly differentiated squamous cells with the most superficial being highly vesiculated with apoptotic nuclei and in the process of desquamating (scale bar=1 μm). B: TEM of the transit-amplifying corneal epithelial cells. These basal cells are large columnar cells and contain large round nuclei with diffuse chromatin (scale bar=1 μm). C: TEM of basal limbal stem cells. These cells are small with irregular nuclei that contain large amounts of condensed chromatin (scale bar=1 μm).

Figure 3

Unstained section of human cornea showing the putative regions where point spectra in transmission mode (4 cm⁻¹ resolution, co-added for 256 scans) were acquired. Black apertures (8 μm×8 μm) designate the stem cell region, blue apertures the transit-amplifying cell region, and green apertures the terminally-differentiated cell region.

Figure 4

Principal component analysis of point mid-infrared spectra over the biologically relevant spectral region (1,800–900 cm⁻¹) derived from the three putative regions (stem cell, transit-amplifying cell, and terminally-differentiated cell). A: The principal component analysis scores plots of stem cell (SC) versus transit-amplifying (TA) cell versus terminally differentiated (TD) cell shows good separation of the three cell populations with a small degree of overlap. B: The resultant loadings plot identifies the biomarker differences (i.e., discriminating wavenumbers) over the spectral range.

Figure 5

Two-class discrimination using principal component analysis over the biologically relevant spectral region (1,800–900 cm⁻¹) derived from the three putative regions (stem cell [SC], transit-amplifying [TA] cell, and terminally-differentiated [TD] cell). Principal component analysis scores and loading plots of A and B SCs versus TA cells; C and D SCs versus TD cells; and E and F TA cells versus TD cells. Cell-specific clusters show good two-class discrimination (A,C,E), and the respective loadings plots (B,D,F) demonstrate major biomarker differences along chosen linear coordinates (i.e., principal components [PCs]).

Figure 6

Spectral image maps of infrared absorbance at wavenumbers (cm⁻¹) chosen to highlight the three putative regions (stem cell, transit-amplifying cell, and terminally-differentiated cell) of human cornea. A: An unstained cryosection of corneal limbus from which the maps were taken. The red rectangle shows the precise area from which the mid-infrared (IR) spectral map was acquired. The dotted red line shows the approximate location of the basal limbal stem cells. The green line indicates the superficial epithelium. B: The limbal region immunolabeled with a green fluorescent marker (keratin 15) for corneal SCs. The cell nuclei have been stained with DAPI (4',6-diamidino-2-phenylindole; blue), and the basement membrane has been immunolabeled for laminin (red). C: Mid-IR spectral map showing absorbance for 1,040 cm⁻¹; D: mid-IR spectral map showing absorbance for 1,107 cm⁻¹; E: mid-IR spectral map showing absorbance for 1,225 cm⁻¹; F: mid-IR spectral map showing absorbance for 1,400 cm⁻¹; G: mid-IR spectral map showing absorbance for 1,525 cm⁻¹; H: mid-IR spectral map showing absorbance for 1,558 cm⁻¹; and I: mid-IR spectral map showing absorbance for 1,728 cm⁻¹.

Figure 7

Distribution analysis of mid-infrared spectra derived from the three putative regions (stem cell, transit-amplifying cell, and terminally-differentiated cell) of human cornea. A: Percentage histograms for stem cells (SCs), transit-amplifying (TA) cells, and terminally differentiated (TD) cells×all wavenumbers put together in a three-dimensional form, where selected wavenumbers have been indicated. B: Mid-infrared spectral map showing absorbance for wavenumber 1,080 cm⁻¹; and, C: specific histograms for selected wavenumbers suggested to be associated with marked class discrimination.