Descemet's Membrane Biomimetic Microtopography Differentiates Human Mesenchymal Stem Cells Into Corneal Endothelial-Like Cells

Abstract

Purpose: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells.

Methods: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level.

Results: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins.

Conclusions: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

FIGURE 1.

Detection of rabbit Descemet microtopography and its ability to convert hMSCs into polygonal zonula occludens (ZO-1) and sodium/potassium (Na/K)-ATPase-expressing cells. A, Enucleated rabbit eyes were prepared (a), CECs were completely removed from Descemet membrane (DM). Then, the cornea inclusive of a small sceral ring was cut out of the eye (b) and put upside down into a silicone ring holder (c). B, In comparison to the untreated rabbit cornea (a), peeled DM (b) is free from any CECs. For better visualization, peeled and unpeeled DMs were stained with hemalaun (c, d). C, Surface microtopography of peeled rabbit DM imaged with Alicona microscopy was determined to have a honeycomb pattern. D, hMSCs were cultivated on the control substrate (smooth collagen) (a, b) or on peeled DM (c, d) for 11 days. In contrast to control cells, cultivation on peeled DM induced expression of ZO-1 (c) and Na/K-ATPase (d).

FIGURE 2.

Cultivation of hMSCs on structure 1 induced morphological changes and finally detachment of cell monolayer like constructs expressing zonula occludens 1 (ZO-1) and sodium/potassium (Na/K)-ATPase. A, hMSCs were cultivated either on smooth silicone (control, a) or on Descemet-like topography (DLT) 1 for 6 days (b). Bright field microscopy pictures (a–d) showed fibroblastic-shaped control cells (smooth silicone) (a, c) and that hMSCs cultured on structure 1 (S1) (b, d) adapted polygonal morphology and allowing the contact with up to 6 neighboring cells around. Fluorescence microscopy images of DAPI-stained samples (e, f) show round-shaped nuclei of cells cultivated on structure 1 (f) in contrast to oval-shaped nuclei of control cells (e). B, hMSCs cultivated on structure 1 changed their morphology into spindle-like shape (a). The cells were stained with DAPI (b). Immunofluorescence analysis revealed that these constructs stained positive with anti-ZO-1 (c) and anti-Na/K-ATPase (d). The overlay of DAPI, Zo-1, and Na/K-ATPase is shown in (e). C, hMSCs cultivated on structure 1 built up single-layer cell associations and peeled off the surface after 10–14 days of incubation. (a) and (b) show two different monolayers. D, hMSCs were cultured on smooth silicone (a–d) as a control and on DLT 1 (e–p) for 14 days. The cells were stained with 4′,6-Diamidino-2-phenylindole (DAPI) (a, c, i, m), anti-ZO-1 (b, f, j, n), or anti-Na/K-ATPase (c, g, k, o). The 3 single-layer cell associations of hMSCs cultured on DLT1 showed upregulation of ZO-1 (f, j, n) and Na/K-ATPase (g, k, o) compared with control cells (b, c). The overlay of DAPI, Zo-1, and Na/K-ATPase is shown in d, h, l, and p.

FIGURE 3.

Effect of high cell density on expression of human corneal endothelial typical genes as paired-like homeodomain (PITX), collagen 8 (Col8), zonula occludens 1 (ZO-1), and sodium/potassium (Na/K)-ATPase and the impact of the Descemet-like topography (DLT) to differentiate high-density seeded hMSC into corneal endothelial-like cells. A, hMSCs were incubated at a concentration of 5000 cells/cm² or 3000 cells/mm² on smooth silicone for 24 hours or 24 days. qRT-PCR was performed, and the gene expression levels of ZO-1, Na/K-ATPase, Col8A2, and PITX were compared. Expression was normalized to beta-2 microglobulin (B2M) and polymerase (Pol) R2A or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An example of the data is presented. B, hMSCs were incubated at a concentration of 3000 cells/mm² on smooth (control) or S1 structured silicone for 24 hours or 24 days. Increased expression of ZO-1, Na/K-ATPase, Col8A2, and PITX were observed after cultivation on DLT1 compared with control cells. Expression levels were normalized to that of MHC class I polypeptide-related sequence A (MICA) and PolR2A. C, hMSCs were cultured on smooth or S1 structured silicone for 14 days. The cells were stained with DAPI (a, d, g, j), anti-Col-8 (b, e), or anti-PITX (h, k). Immunofluorescence analysis revealed that DLT induced Col-8 (e) and PITX (k) expression. Figure C c and f demonstrate the overlay of DAPI and Col 8 of Control and Structure and Figure C i and l demonstrate the overlay of DAPI and PITX of Control and Structure. Control cells minimally expressed Col-8 (b) but no PITX (h).

FIGURE 4.

Impact of biomimetic collagen to enhance expression of corneal endothelial cell (CEC)-typical markers. A, hMSCs were incubated at a seeding density of 3000 cells/mm² on smooth silicone or on smooth collagen for 5 days. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed. Gene expression of zonula occludens 1 (ZO-1), sodium/potassium (Na/K)-ATPase, collagen 8 (Col-8), and paired-like homeodomain (PITX) was clearly increased after cultivation on smooth collagen as compared to smooth silicone. Expression levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). B, hMSCs were incubated at a seeding density of 3000 cells/mm² on smooth or DLT1 structured collagen for 7 days. Gene expression of ZO-1, Col-8, and PITX was clearly increased after cultivation on Descemet-like topography (DLT1) collagen as compared to smooth collagen. Expression levels were normalized to those of GAPDH. C, hMSCs were cultured on smooth or DLT structured collagen for 14 days. Cells were stained with DAPI (a, b), anti-ZO-1 (c, d), and anti-Na/K-ATPase (e, f). Immunofluorescence analysis revealed that DLT induced ZO-1 (D) and Na/K-ATPase (f) expression. Control cells (c, e) expressed also both markers to a minor degree. The overlay of Dapi and ZO-1 or Na/K-ATPase is shown in g and h.