Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells

Abstract

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.

Keywords: Cornea; Expansion; Isolation; Laminin; Limbal epithelial progenitor cells; Limbal stem cells.

Figure 1.. Expression of laminin chains in the limbal stem cell niche in situ.

A. Quantitative real-time polymerase chain reaction (qRT-PCR) primer assays showing higher expression levels of laminin α2 (LAMA2), α4 (LAMA4), α5 (LAMA5), β2 (LAMB2), β3 (LAMB3), and γ2 (LAMC2) in microdissected limbal epithelial stem/progenitor cell (LEPC) clusters compared with basal corneal epithelial cell (BCEC) populations; laminin α1 (LAMA1), α3 (LAMA3), β1 (LAMB1), β4 (LAMB4), γ1 (LAMC1) showed no differential expression patterns. B. Immunofluorescence analyses of corneoscleral tissue sections showing differential staining patterns of laminin α2, α5, β2, β3, γ2, and γ3, but similar staining patterns of laminin α1, α3, β1, and γ1 in the basement membranes of corneal and limbal epithelia; laminin α4 was largely negative in epithelial basement membranes. C. Immunofluorescence double labeling of laminin (LN) α5 (green) and cytokeratin (CK)15, N-Cadherin, p63α, integrin α6, integrin α3, and integrin β1 (red); nuclear counterstaining with DAPI (blue); scale bar  =  20 µm. Reprinted from Polisetti et al., 2017 , licensed under a CC BY 4.0.

Video 1.. Limbal Epithelial Progenitor Cell Isolation

Figure 2.. Isolation of limbal epithelial progenitor cells.

A. The corneal scleral rim (left) was cut into sectors and each sector was trimmed off 1mm before and after the limbal region (right). B. Different sizes of limbal clusters and single cells (left) formed after overnight incubation of limbal segments in collagenase solution (x40 magnification). Limbal cluster-derived limbal epithelial cells cultured in KSFM media at 50% confluence (right, top) and 100% confluence (right, bottom) (x40 magnification).