Abstract
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.
Highlights
Limbal epithelial stem/progenitor cells (LEPC) are located at a specific anatomic location referred to as the limbal stem cell niche and regulate homeostasis of the corneal epithelium (Gonzalez G et al, 2018)
Double immunostaining confirmed the co-localization of CD90 and vimentin in sub-epithelial stromal cells, which were in close association with basal layers of limbal epithelium, as well as blood vessels of the limbal stroma (Figure 1B, yellow arrows)
Immunostaining of cultured limbal clusters (Figure 1C) derived from collagenase digestion showed the expression of keratins (pan-cytokeratin (PCK), green) and vimentin in epithelial cells; Melan-A and vimentin expression in melanocytes, which are interspersed between the epithelial cells and vimentin expression in stromal cells
Summary
Limbal epithelial stem/progenitor cells (LEPC) are located at a specific anatomic location referred to as the limbal stem cell niche and regulate homeostasis of the corneal epithelium (Gonzalez G et al, 2018). The limbal stem cell niche is characterized by a specific extracellular matrix (ECM) composition, limbal vasculature, and surrounding limbal niche cells (LNCs) [1–3]. LNCs mainly comprise intraepithelial melanocytes (limbal melanocytes, LM) and subepithelial stromal cells (limbal mesenchymal stromal cells, LMSC), which have been shown to support the corneal epithelial regeneration during wound healing and maintenance of LEPC phenotype both in vitro and in vivo [3,5–9]. The co-cultivation of LEPC with LMSC/LM could represent an improved strategy to generate cell transplants for patients suffering from limbal stem cell deficiency [10,11]. Both LMSC and LM were shown to have potent immunomodulatory, anti-inflammatory and anti-angiogenic properties, making them attractive tools for clinical use [9,12–14]. Research on freshly isolated LMSC or LM, which may closely resemble the LNC in vivo, has been hampered by the lack of an efficient and fast protocol for isolating these cells as pure populations
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