Existence of Corneal Endothelial Slow-Cycling Cells.

The corneal endothelium is a monolayer of hexagonal cells derived from the neural crest that is responsible for maintaining the appropriate degree of corneal hydration necessary for vision. The normal endothelial cell count is 2 to 5 × 103 cells/mm2 in humans.1 An endothelial cell count below a threshold of 300 to 500 cells per mm2 is associated with irreversible corneal swelling, epithelial blistering, loss of vision, and need of transplantation.1,2 The current dogma is that mature corneal endothelial cells are quiescent, arrested in the G1-phase of the cell cycle, with limited proliferative capacity in vivo.3–5 Cell contact inhibition has been proposed to have a major role inhibiting endothelial replication.3 However, corneal endothelial cells can replicate in cell cultures and tissue explants. In addition, the presence of endothelial cells expressing progenitor cell markers in the human corneal periphery suggest that endothelial cells or at least some endothelial cells may replicate or have the ability to replicate in an appropriate environment.6–8 Two different facts strongly suggest that human endothelial cells proliferate in vivo and repopulate donor grafts. First, studies in sex-mismatched transplanted corneas showed the presence of a mixture of donor and recipient endothelial cells in the transplanted graft.9,10 Endothelial replacement by the host cells in failed edematous corneas, and also in cases where corneas were clear was demonstrated suggesting that endothelial replacement occurs in failed and successful transplants.9 Second, anecdotal clinical reports show that human endothelial cells can replicate in vivo in the periphery of the posterior cornea and migrate and restore Descemet's membrane, and the endothelial monolayer integrity reestablishing corneal transparency.11,12 Nestin, a class VI intermediate filament, has been used to identify cells with progenitor cell properties in the developing neural cortex,13 cultured primary neurons from the central and peripheral nervous systems,14 immortalized cell lines, and tumors.15 It is well documented that expression of nestin reflects the undifferentiated state of neural progenitor cells16 and that downregulation of nestin reactivity is associated with the differentiation of neural progenitor cells in the developing nervous system.16 Corneal keratocytes and endothelial cells originate from multipotent cranial neural crest cells that migrate rostrally to form facial structures.17 The low-affinity nerve growth factor receptor (NGFR) is one of the two receptor types for the neurotrophins and has been used as a neural crest stem cell marker.18–20 A pure or enriched population of neural crest stem cells has been isolated from mouse trunk neural tubes and embryonic peripheral nerves using low-affinity NGFR as a marker.18,19 These cells that are NGFR-positive had self-renewal capacity, and displayed multipotent differentiation properties. Other progenitor cell markers include Sox9 and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5). Sex-determining region Y–box containing gene 9 (Sox9), high-mobility group box (Sox) transcription factors, is mutated in campomelic dysplasia, a disorder characterized by skeletal malformations, XY sex reversal, and neonatal lethality.21 Sex-determining region Y–box 9 is expressed during embryogenesis in several tissues and organs, including the central nervous system.22 Initially identified as a Wnt target gene in human colon cancer cell lines harboring Wnt-activating mutations,23 Lgr5 is a well-established epithelial intestinal stem cell marker.24,25 Detection of Ki-67 is commonly used in histological specimens to detect cell proliferation and provides an index of proliferation with prognostic value.26 It was initially reported in the early 1980s.27 It is a nonhistone nuclear and nucleolar protein strictly associated with cell proliferation and is detected during all the active phases of the cell cycle, but is absent in resting cells. Its strict association with cell proliferation and its coexpression with other well-known markers of proliferation indicate a pivotal role in cell division. Expression of Ki-67 is upregulated in the G2, M, and latter half of the S phase of the cell cycle.28 Progenitor cells are able to differentiate into one or more cell types, but do not have unlimited differentiation or replication properties like stem cells. Progenitor cells can adjust their cell-cycle properties, in homeostasis or during injury, to the circumstances that surround them.29 A classic way to search for stem cells or progenitors cell niches is the use of labeling agents like 5-bromo-2′-deoxyuridine (BrdU).29 Slow-dividing cells found in hair follicles and the corneal limbus have been previously described as stem cells.30 Therefore, this slow-cycling characteristic is considered a common feature to identify stem cells in different tissues and organs. Here we demonstrate the presence of populations of progenitor cells in the corneal periphery as well as the existence of slow-cycling cells in the extreme periphery of the corneal endothelium.