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 crest-derived 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 cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2β), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, β-III 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.

Supplemental Figure S1

Specular image of 69-year-old man with Fuchs corneal endothelial dystrophy demonstrating stage 3⁺ guttae. AVE, average; CD, cell density; CV, coefficient of variation; HEX, hexagonal cell ratio; MAX, maximum; MIN, minimum; NUM, number; OD, right eye.

Supplemental Figure S2

Differentiated neuronal markers GFAP, MAP2, and Tubulin-3 are negative in undifferentiated HCEnCs of all three cell types. Bottom panels show differentiated neural crest–derived progenitor cells isotype controls for GFAP, MAP2, and β III-Tubulin. GRAP, glial fibrillary acidic protein; HCEnC, human corneal endothelial cell; MAP2, microtubule-associated protein 2.

Supplemental Figure S3

Expression of ZO-1 (differentiated marker for corneal endothelial cells) in differentiated neural crest–derived progenitor cells from normal and Fuchs donor by differentiation medium (Chen's medium). HCEnC, human corneal endothelial cell; PI, propidium iodide.

Figure 1

Ex vivo expansion of normal HCEnCs harbors progenitor-like subpopulation with distinct growth characteristics. A–C: Phase-contrast micrographs of two morphologically distinct subpopulations of highly proliferative HCEnCs (black arrows) and slow proliferating fibroblast-like HCEnCs (white arrows) generated from three normal donor corneal specimens from men 21 years (A), 56 years (B), and 70 years (C) of age. D–I: Phase-contrast micrographs of the highly proliferative subpopulation within HCEnCs at early passage (D), 56M (E), and 70M (F) and late passage (G), 56M (H), 70M (I). J–O: Phase-contrast micrographs of spheres formed by HCEnCs at 7 days after seeding in SF medium (J–L) and leading edge of the spheres from three normal donor HCEnCs at day 7 on 1% Matrigel (M–O). Scale bars = 100 μm (A–O). HCEnC, human corneal endothelial cell; SF, sphere formation.

Figure 2

Elevated expression of neural crest–derived progenitor markers in FECD and normal HCEnCs compared with slow proliferating cells. A: Phase-contrast micrograph showing two morphologically distinct subpopulations of cells (white arrow, fibroblast-like cells with reduced proliferation; black arrow, rapidly proliferating cells), cultured from FECD ex vivo specimen (69-year-old man, designated as F-HCEnC-69M). B: Rapidly proliferating progenitor subpopulations of cells separated from slow dividing primary cells shown in A at 3 days after seeding. C: Progenitor population shown in B was trypsinized at day 6 and cultured for 5 days. D and E: Phase-contrast micrographs of sphere formation (D) and leading edge of sphere (E) by F-HCEnC-69M at passage 10. F–J: Gene expression analysis of neural crest–derived progenitor cell markers (PAX3, SOX9, AP-2β, Nestin, and p75^NTR) by real-time PCR in early and late passage HCEnCs derived from three normal donors and one FECD specimen. The relative expression of all markers in each cell type was compared with the mean expression of two normal and one FECD slow dividing primary cells. The early (passage 10 to 15) and late (passage 70 to 75) samples, except the early passage FECD samples which were derived between passages 10 to 16. RT-PCR shows significantly higher expression in HCEnCs at early and late passages than primary HCEnC, PAX3, Nestin, SOX9, and AP-2β. Data are expressed as means ± SEM. n = 3 experiments. ^∗P < 0.05, ^∗∗∗P < 0.001 by one-way analysis of variance. Scale bars = 100 μm (A–E). FECD, Fuchs endothelial corneal dystrophy; HCEnC, human corneal endothelial cell.

Figure 3

Cell proliferation and CFE of normal and FECD-derived progenitors is retained with age and passaging. A: CDT was calculated by seeding 50,000 cells per well in 12-well plates and counted after 2, 3, and 4 days using a Countess Cell Counter. Graph represents the CDT for early (10 to 15) and late (68 to 72) passages of HCEnC-21M, early (10 to 15) and late (70 to 75) passages of HCEnC-70M and early (9 to 14) and late (67 to 74) passages. HCEnC-56M and -70M show a higher proliferation rate at early passages than late passages; however, no significant difference is observed in proliferation of HCEnC-21M at higher passage. Primary corneal endothelial cells show a significantly slower proliferation. B: F-HCEnC-69M (passage 10 to 13) shows a higher proliferation rate with CDT of 32 hours than the slow proliferating cells from Fuchs tissue. C: The CFE of all three corneal endothelial progenitors quantified at earlier (10 to 14) and late (65 to 75) passages shows an age- and passage-dependent reduction in their colony forming ability. D: Quantification and CFE of early passage (10 to 13) F-HCEnC-69M shows an increased efficacy compared with the primary cells derived from FECD specimen. Data are expressed as means ± SEM. n = 4 (C); n = 3 experiments (A, B, and D). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 by one-way analysis of variance. CDT, cell doubling time; CFE, colony forming efficacy; FECD, Fuchs endothelial corneal dystrophy; HCEnC, human corneal endothelial cell.

Figure 4

Normal donor- and FECD specimen-derived progenitors express characteristic pluripotent markers. A–D: Spheres were generated from NCDPs on a 1% Matrigel-coated surface and tested for differential expression of stem cell markers. Relative expression of p63, OCT4, LGR5, and SOX2 are significantly higher in all progenitors irrespective of age and passage than nondividing primary HCEnCs. E: Spheres generated from F-HCEnC-69M (passage 15 to 24) show significantly higher relative expression of OCT4, SOX2, LGR5, and p63 than Fuchs nondividing cells (relative expression was below C_T). F and G: Spheres generated from early (F) and late (G) passage normal donor NCDPs were stained for SSEA4, TRA-1-60, LGR5, and OCT4. The expression of SSEA4 and TRA-1-60 decreases with passage, whereas both LGR5 and OCT4 show a reduced expression in HCEnC-56M and -70M. H: Spheres generated from early passage F-HCEnC-69M cells express stem cell markers SSEA4 and OCT4 similar to early passage HCEnC-56M and -70M. TRA-1-60 shows a reduced expression compared with all three cell types in F. LGR5 shows an intracellular localization that differs from HCEnC-21M and -56M. Data are expressed as means ± SEM. n = 6 experiments (A–E). ^∗P < 0.05, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001 by two-tailed Student's t-test for F-HCEnC-69M and one-way analysis of variance for the others. Scale bars = 100 μm. FECD, Fuchs endothelial corneal dystrophy; HCEnC, human corneal endothelial cell; NCDP, neural crest–derived progenitor; ND, not detected.

Figure 5

Normal and FECD-derived progenitors can undergo differentiation to neuronal lineage. Late-passage NCDPs generated after trypsinization of spheres were seeded at a density of 2.5 × 10⁴ cells/cm² on glass coverslips coated with 100 μg/mL poly-d lysine and 20 μg/mL laminin. Cells were differentiated with neural induction media for 7 days, and immunofluorescence was performed for GFAP, MAP2, and β-III tubulin. HCEnC-21M, 56M, 70M, and F-HCEnC-69M express neuronal markers suggestive of differentiation to neuronal lineage. Scale bars = 100 μm. FECD, Fuchs endothelial corneal dystrophy; GFAP, glial fibrillary acidic protein; HCEnC, human corneal endothelial cell; MAP2, microtubule-associated protein 2; NCDP, neural crest–derived progenitor; PI, propidium iodide.

Figure 6

NCDPs differentiate into corneal endothelium that retains p75-positivity in vitro. A: Spheres generated from early and late-passage NCDP cells were trypsinized and cultured on FNC-coated coverslips and allowed to differentiate for 6 days in Chen's medium. All NCDP cells express the neural crest marker p75^NTR and Na⁺/K⁺ ATPase that labels the corneal endothelium. Nondividing primary HCEnC were negative for p75^NTR. B: NCDP cells derived from spheres were plated in 12-well transwell inserts (0.4 mm) at a density of 100,000 cells per transwell, and TEnR was measured every 4 days for 20 days. Note that HCEnC-21M, -56M, -70M, and F-HCEnC-69M establishes a typical corneal endothelial barrier that ranges between 10 and 13 Ω cm² at 12 days that continues until 20 days. Previously established corneal endothelial cell line (HCEnC-21T) was used as positive control. The TEnR value for each time point represents the average of experiments. Data are expressed as means ± SEM. n = 6 experiments (A); n = 3 independent experiments with each experiment performed in duplicate (B). Scale bars = 100 μm. HCEnC, human corneal endothelial cell; NCDP, neural crest–derived progenitor; PI, propidium iodide; TEnR, transendothelial resistance.

Figure 7

Diagram of the role of the adult NCDPs in endothelial cell renewal. Highly proliferative colonies isolated from normal and FECD corneal endothelium demonstrate expression of neural crest progenitor markers and propensity for neurosphere formation which fosters stem cell niche in vitro and promotes differentiation into neuronal structures and p75-positive differentiated endothelial cells, indicating a potential application for future regenerative therapies. FECD, Fuchs endothelial corneal dystrophy; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; NCDP, neural crest–derived progenitor.