Localisation of Epithelial Cells Capable of Holoclone Formation <i>In Vitro</i> and Direct Interaction with Stromal Cells in the Native Human Limbal Crypt

<div><p>Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used <i>in vitro</i> clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.</p></div

Cell-to-cell contacts imaged within the limbal crypts by SBFSEM.

<p>Box in A and F represents the area in which contact between putative LESC (green arrows in B, C, G, H and green volumes in D, E, I, J after 3D reconstruction) and limbal stromal cells (yellow arrows in B, C, G, H and yellow volumes in D, E, I, J after 3D reconstruction) was observed. Cell nuclei are represented in pink in D an E, the area of interaction between putative LESC and the limbal stromal cell is represented in orange (J). Scale bars: 50 μm (A, F); 5 μm (B, C, G, H).</p

Limbal crypt ultrastructure observed by SBFSEM.

<p>Limbal crypt tangentially imaged through 70 μm from the corneal to the conjunctival side of the limbus. A, B and C represent non-sequential micrographs of the same 3D dataset. Manual segmentation followed by 3D reconstruction highlights the close proximity between the limbal epithelium (yellow volume in D) and a limbal stromal cell (white asterisks in B, blue area in C and blue volume in D) within the limbal crypt (Lc) suggesting a putative cell-to-cell contact. Lc: limbal crypt; Bv: blood vessel; St: Stroma. Arrowheads: melanocytes. Scale bars: 50 μm.</p

Observation of putative LESCs by TEM within the LCs.

<p>Transmission electron micrographs highlighting the interface between the limbal stroma and the limbal basal epithelial layer within the non-crypt rich limbus (A, B) and the limbal crypts (C, D, E, F). White arrowheads: hemidesmosomes; Black arrows: limbal stromal cell extensions in the vicinity of putative LESCs. Epi.: epithelium; St.: stroma; Bm: Basement membrane. White asterisks: putative LESCs. Black arrowhead: Contact between putative LESC and limbal stromal cell. Black box in E represents area in F. Scale bars A, B, C, D and E: 2 μm, F: 1 μm.</p

Clonal analysis.

<p>Crypt-rich (A) and non-crypt rich (D) limbal biopsies observed under the dissecting microscope and targeted for clonal analysis. Sections were cut tangentially to the limbal circumference and stained with hematoxylin and eosin. Sections shown highlight the palisades of Vogt and the limbal crypts (B) and the non-crypt rich limbus (E). Colonies of limbal epithelial cells grown in Petri dishes and stained with 2% rhodamine (C) and (F). Growth potential of single epithelial cells isolated from crypt-rich (C) and non-crypt rich (F) human limbal biopsies was characterized by the generation of holoclones, meroclones and paraclones. LECs isolated from the limbal crypts generated the highest proportion of holoclones demonstrating their stem characteristics and the LCs as a stem cell niche. Scale bars: 50 μm. Black arrows: Limbal crypts. Conj.: conjunctiva; L: Limbus; CC: central cornea; Epi.: epithelium; St.: stroma; POV: palisades of Vogt; LC: limbal crypt.</p

Melanocytes interact with putative LESCs in their niche.

<p>Immunohistochemistry reveals much more MelanA positive cells within the LCs than within the non-crypt rich limbal areas (A, B). Serial-block face scanning electron micrographs showing the edge of one limbal crypt (C, D). After manual segmentation and 3D reconstruction (E, F, G) putative LESC (smallest epithelial cell with a high nucleus/cytoplasm ratio) is represented in green, melanocyte (dendritic cell containing electron dense granules) in red, limbal stromal cell in yellow. Blue volumes correspond to nuclei. Continuous line in G corresponds to block face represented in C. Dashed line in G corresponds to block face represented in D. Scale bars: 50 μm (A, B) and 7 μm (C, D).</p

Immunochemical analysis of primary hCECs on RAFT.

<p>Primary hCECs seeded on glass slides and fixed after 4 days, stained with (A) ZO-1 or (B) Na⁺/K⁺-ATPase (green) counterstained with DAPI (blue). Primary hCECs seeded onto RAFT stained with either (C and E) ZO-1, (D and F) Na⁺/K⁺-ATPase (green) counterstained with DAPI (blue). (C and D) fixed after 4 days in culture or (E and F) after 14 days. Scale bars 50 µm.</p

Immunochemical analysis of hCECL cells on RAFT.

<p>hCECL seeded on RAFT (A) at 3000 cells/mm² with C/L coating or (B) 3000 cells/mm² on FNC coating stained with ZO-1 (green) and counterstained with propidium iodide (red). hCECL seeded on RAFT (C) at 2000 cells/mm² with C/L coating or (D) seeded at 4000 cells/mm² on FNC coating stained with Na⁺/K⁺-ATPase (green) and counterstained with propidium iodide. (E) Negative isotype control. (F) hCECL on permanox slides with FNC coating stained with Na⁺/K⁺-ATPase (green) and counterstained with propidium iodide. Scale bars 50 µm.</p

Scanning electron microscope characterisation of RAFT with hCECs.

<p>(A) Representative micrograph of the surface of RAFT constructs. (B) Representative low magnification SEM image showing confluent monolayer of hCECs on RAFT. (C) Higher magnification image showing cell borders between cells and (D) at high magnification over-lapping finger-like projections onto juxtaposed cells. Scale bars A 5 µm, B 50 µm, C 10 µm, D 2 µm.</p

Loading and insertion of RAFT into an <i>ex vivo</i> porcine eye using Tan EndoGlide™.

<p>Representative photographs showing the process of loading (A–D) of the Tan EndoGlide™ with RAFT construct and insertion (E–H) of RAFT into the anterior chamber of an <i>ex vivo</i> porcine eye model. (A) Loading forceps grasp the edge of the RAFT construct from the spatula. (B) RAFT is pulled into the cassette and (C) automatically coils into a double coil configuration. (D) RAFT is fully loaded into the cassette with no upper surfaces touching. (E) Tan EndoGlide ™ is inserted into the anterior chamber that is prevented from collapsing using a column of saline via an inserted needle. (F) RAFT is pulled from the cassette (G) into the anterior chamber and positioned centrally before (H) an air bubble is inserted to appose RAFT to the posterior cornea.</p