Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus

The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117(-)CD90(+)), LM (CD117(+)CD90(-)), and LEPC (CD117(-)CD90(-)). The LMSC exhibited self-renewal capacity (55.0 +/- 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4-5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63(+) and Ki67(+) cells and decreased CK12(+) cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.

Automated summary

The fate of limbal epithelial progenitor cells (LEPC) in the corneal limbus of humans depends on the microenvironment, particularly limbal niche cells (LNC). However, research on LNC has been limited by a lack of effective protocols to isolate and purify these cells. In this study, we developed a protocol to retrieve pure populations of LNC, including limbal mesenchymal stromal cells (LMSC), limbal melanocytes (LM), and LEPC, using collagenase digestion and fluorescence-activated cell sorting (FACS). The sorted cells were characterized and the effects of LMSC and LM on LEPC were investigated in 3D co-cultures. The results showed that LMSC exhibited self-renewal capacity and expressed mesenchymal stem cell markers, while LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers and showed colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a stratified epithelium and preserved the LEPC phenotype. This efficient isolation protocol allows for further transcriptomic, proteomic, and functional studies on LNC, providing valuable insights into the limbal stem cell niche.