Existence of Neural Crest–Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium

Abstract

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crestederived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cellerelated genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75 NTR ), PAX3, SOX9, AP2B1 (AP-2b), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, beIII tubulin, and glial fibrillary acidic protein positive neurons and into p75 NTR -positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells on the posterior surface of the cornea and are essential for maintaining appropriate corneal hydration necessary for clear vision. HCEnCs sustain corneal clarity by serving as a barrier between the aqueous humor and the corneal stroma and by active ionic transport that regulates the swelling pressure of the cornea. HCEnCs are arrested in the postmitotic state and have limited proliferative capacity both in vivo and in vitro. The postmitotic arrest has been attributed to contact inhibition, transforming growth factor-b in aqueous fluid, and lack of paracrine stimulation by growth factors of cell-cycle promotion from the G 1 to the S phase.1 In a normal human life span, the endothelial cell density gradually declines.2 Therefore, age-and disease-related HCEnC loss is a major cause of corneal blindness requiring corneal transplantation to restore vision. Specifically, Fuchs endothelial corneal dystrophy (FECD) is the most common cause of endogenous endothelial cell dysfunction; it is associated with progressive cell apoptosis and concurrent extracellular matrix deposition in the form of guttae and is primarily treated by allogeneic endothelial keratoplasty.3,4 The pathogenic mechanism behind FECD is purportedly related to the interplay between genetic mutations and environmental factors. 5Several reports have proposed the existence of endothelial progenitor cells situated in the peripheral cornea, but evidence so far has not been conclusive. 6e8 The description of purported stem cells (SCs) has been based on ex vivo

A review of the evidence for in vivo corneal endothelial regeneration

“…Most interestingly, these characteristics were determined not only on human corneal endothelial cells in an early and late passage but also on cells derived from FECD patients. 60 These results are suggestive, but although it is tempting to think that there may be an endothelial stem cell population, without human studies using cellular labeling in vivo, it is difficult to conclude with certainty if the corneal endothelial cells are proliferating or just enlarging and migrating.…”

“…82 Although neural crestederived progenitor cells also can be isolated from FECD eyes, the question remains whether the migrated and/or proliferated cells can function and, if so, for how long? 60 Currently, endothelial cell loss 1 year after conventional EK ranges between 30% and 50%, largely as a result of graft preparation and surgical manipulation. 6,12,66,70,94,117 A 5-year follow-up study revealed a mean loss of 53% of endothelial cells after EK, considerably lower than the 70% seen with PK, while graft survival was similar.…”

Endothelial–Stromal Communication in Murine and Human Corneas

“…An enrichment of p75NTR (neurotrophin receptor) positive cells at the corneal endothelial side of the transition region between the corneal endothelium and the trabecular meshwork has been shown; however, no statistically significant difference was found in colony forming efficiency or number of p75NTR positive cells (Hara et al 2014). The presence of less differentiated cells and expression of stem cell markers in the extreme corneal endothelial periphery also has been described (He et al 2012; Katikireddy et al 2016). Our laboratory reported the presence of slow cycling cells in the murine endothelial extreme periphery (Espana et al 2015).…”

A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

“…Nevertheless, this remains a great challenge as CEC have a restricted proliferative ability in humans, limiting its feasibility [4]. Furthermore, donor corneas are generally from older patients and these cells present even lower proliferation rates, being more prone to senescence than younger corneas [5][6][7][8]. In order to overcome the senescence state of cells, a recent study cultured CEC from a young donor and injected them with ROCK inhibitor, which has been described to reduce intraocular pressure, to increase CEC cell density and to promote the typical polygonal monolayer phenotype [9,10].…”