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(Investigative Ophthalmology and Visual Science. 2005;46:3645-3648.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0630
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Comparison of Rabbit Corneal Endothelial Cell Precursors in the Central and Peripheral Cornea

Tatsuya Mimura,1 Satoru Yamagami,2 Seiichi Yokoo,2 Makoto Araie,1 and Shiro Amano1

1From the Departments of Ophthalmology and 2Corneal Tissue Regeneration, University of Tokyo Graduate School of Medicine, Tokyo, Japan.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To compare the distribution and self-renewal capacity of rabbit corneal endothelial cell precursors in the central and peripheral regions of the cornea.

METHODS. The corneal endothelium (CE) and Descemet’s membrane of New Zealand White rabbit corneas were divided into a peripheral region (6.0–10.0 mm in diameter) and a central region (6.0 mm in diameter). Then a sphere-forming assay was performed to isolate precursors from the CE of each region. Numbers of primary and secondary sphere colonies and sizes of primary spheres were compared between the central and peripheral regions.

RESULTS. Primary spheres were isolated from the peripheral and the central regions of the CE. The rate of primary sphere formation in the peripheral region (34.4 ± 10.4/10,000 cells) was significantly higher than in the central cornea (26.8 ± 6.6/10,000 cells; P = 0.0042), but there was no significant difference in the size of primary spheres between the two regions. Self-renewal capacity was higher in the peripheral region than in the central region, as evidenced by a significantly higher secondary sphere formation rate for cells from the periphery (39.0 ± 8.8/10,000 cells) compared with that for cells from the central region (25.4 ± 4.2/10,000 cells; P = 0.00028).

CONCLUSIONS. These findings demonstrate that peripheral and central rabbit corneal epithelia contain a significant number of precursors but that the peripheral endothelium contains more precursors and has a stronger self-renewal capacity than the central region.

The corneal endothelium (CE) is derived from the neural crest1 2 and forms a single layer of hexagonal cells. In the human CE, wound healing occurs predominantly by cell migration and enlargement without discernible cell proliferation.3 4 In various tissues that show vigorous proliferative activity, the existence of adult stem cells or precursors is predictable. However, the human CE shows weak to no proliferative capacity in vivo, unlike the results for cultured cells.5 6 7 Hence, little attention has been directed to the investigation of adult stem cells or precursors of CE.

The sphere-forming assay has been widely used to isolate adult stem cells or precursors.8 9 10 11 12 13 14 15 16 17 18 19 20 If dissociated single cells from each tissue form cell clusters (spheres) under serum-free conditions and the spheres and their progenies show respective self-renewal and multipotential capacity, the spheres should contain adult stem cells or precursors. Using this method, the existence of adult stem cells or precursors has been reported in various tissues, including the central nervous system,8 9 bone marrow,10 skin,11 12 inner ear,13 retina,14 15 pancreas,16 corneal limbal explants,17 and corneal stroma.18 We isolated precursors from human and rabbit CE by the sphere-forming assay and showed that these CE-derived precursors could have a propensity to differentiate into corneal endothelial cell-like cells that produced neuronal and mesenchymal proteins.19 20

The proliferative activity and density of cells at the peripheral and central regions of the human cornea are controversial. In vitro, peripheral CE has a higher mitotic activity than CE from the central region.21 The cornea has a higher density of endothelial cells in the peripheral region of the CE than in the central region.22 23 p53, involved in the negative regulation of cell division, is more highly expressed in the central CE.24 Moreover, BrdU incorporation in unwounded CE and telomerase activity were detected in CE limbal area only (Whikehart DR, et al. IOVS 2002;43:ARVO E-Abstract 1627; Whikehart DR, et al. IOVS 2004;45:ARVO E-Abstract 3765). On the other hand, Konomi and Joyce (Konomi K, et al. IOVS 2005;46:ARVO E-Abstract 4710) reported that there was no significant difference in the proliferative capacity of human CE between the central and the peripheral regions. The density of peripheral CE was reported to be lower than that of the central CE in humans.25 Therefore, clarification of the distribution of CE precursors may provide some clues about the proliferative capacity and density of the CE at the center and the periphery.

In this study, we investigated the distribution and proliferative capacity of rabbit CE precursors from the central and peripheral regions by a sphere-forming assay. We used rabbit corneas because the distribution of the CE can be examined at a younger age after birth.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Primary Sphere-Forming Assay and Sizes of Spheres from Peripheral and Central CE
Rabbits were treated in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. CE and Descemet’s membrane were separated from the periphery (6.0–10.0 mm in diameter) and from the central area (6.0 mm in diameter) in New Zealand White rabbits (15–18 weeks old; Saitama Experimental Animals Inc., Saitama, Japan) using forceps and were incubated for 3 hours at 37°C in basal medium containing 0.02% collagenase (Sigma, St Louis, MO). Next, they were incubated in 0.2% EDTA at 37°C for 5 minutes and then were dissociated into single cells by trituration with a fire-polished Pasteur pipette. Viability of the isolated CE cells was greater than 90%, as shown by trypan blue staining (Wako Pure Chemical Industries, Osaka, Japan). The sphere-forming assay was used for primary cell culture.8 Basal medium containing a methylcellulose gel matrix (0.8%; Wako Pure Chemical Industries) was used to prevent cell reaggregation, as described previously.26 27 Cells were plated at a density of 10 viable cells/ìL (50,000 cells/well; 2500 cells/cm2) in the uncoated wells of 60-mm culture dishes. The basal medium was Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with B27, epidermal growth factor (EGF; 20 ng/mL), and basic fibroblast growth factor (bFGF; 40 ng/mL). The number of spheres per 10,000 cells was calculated for each well. To measure sphere colony diameters, cultures were observed under an inverted phase-contrast microscope (ELWD 0.3; Nikon, Tokyo, Japan) with a 10x objective lens, and images were analyzed using the NIH image program developed at the US National Institutes of Health (n = 10).

Secondary Sphere Formation from Primary Spheres Derived from Peripheral and Central CE
For passaging, primary spheres (day 7) were treated with 0.5% EDTA and were dissociated into single cells plated into 60-mm culture dishes at a density of 10 cells/µL. Culture was continued for 7 days in basal medium containing methylcellulose gel matrix to prevent reaggregation. These experiments were performed twice; representative results are shown (n = 10) (see Fig. 3 ).



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  		FIGURE 3. Formation of secondary spheres from primary spheres derived from the periphery and center of the cornea. Secondary spheres were generated from dissociated primary spheres. (A) Replating efficiency of the secondary spheres is higher with cells from the peripheral CE (n = 10) than from the central CE (n = 10) (unpaired t test). Representative photographs of secondary spheres derived from the peripheral (B) and central (C) regions of the cornea are shown. Scale bar, 100 µm. The experiment was repeated twice with different sets of animals, and representative data are shown. *P = 0.00028.

 
Statistical Analysis
The unpaired t test was used to compare mean values. Significance was set at P < 0.05, and all analyses were performed using a statistical software package (StatView Version 5; Abacus Concepts, Berkeley, CA).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Primary Sphere Formation and Sphere Size
The CE was disaggregated into single cells cultured for 7 days. During that period, the spheres grew larger, whereas nonproliferating cells were eliminated. To compare the densities of precursors between peripheral and central regions, primary spheres were isolated from the peripheral and the central CE. Photographs of representative spheres from the periphery and the center are shown in Figure 1A . When the number of sphere colonies obtained was counted, a significantly higher number of spheres (34.2 ± 10.4/10,000 cells, mean ± SD) was obtained from the peripheral region of the CE compared with the central region (26.8 ± 6.6) per 10,000 cells (Fig. 1B) . No statistically significant difference was noted with respect to the size of primary spheres from the two regions after 3, 5, and 7 days, indicative of no difference of proliferative capacity in spheres derived from each region (Fig. 2) .



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  		FIGURE 1. Primary sphere formation by rabbit CE from the periphery and center of the cornea. Representative photographs of primary spheres from the periphery (A) and center (B) of the cornea are shown. (C) The number of sphere colonies obtained from the peripheral region (n = 10) is significantly higher than from the central region (n = 10) after 7 days of culture (unpaired t test). The experiment was repeated twice from different sets of animals, and representative data are shown. *P = 0.0042. Scale bar, 50 µm.

 


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  		FIGURE 2. Comparison of the sizes of primary spheres from the periphery and center of the cornea. Sizes of primary spheres derived from the peripheral and central areas of the cornea were measured (n = 10). The mean size of spheres from both areas gradually increases during culture (229 ± 53 µm vs. 192 ± 64 µm after 7 days, respectively). The experiment was repeated twice, and representative data are shown. n.s., not significant.

 
Secondary Sphere Formation
To evaluate the self-renewal capacity of the CE, primary spheres were passaged under the same culture conditions as those for the growth of primary spheres. Secondary spheres were generated from dissociated primary spheres derived from the peripheral or the central CE. The number of secondary spheres per 10,000 cells was significantly higher with spheres derived from the peripheral region than from the central region (39.0 ± 8.8 vs. 25.4 ± 4.2, respectively; P = 0.00,028; unpaired t test; Fig. 3A ). Photographs of representative secondary spheres are shown in Figure 3B .


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that spheres derived from rabbit CE have a high proliferative capacity and that their progeny express a mesenchymal marker ({alpha}-smooth muscle actin) and neuronal lineage markers (neuron-specific enolase and microtubule-associated protein-2).20 This study further demonstrated that peripheral and central regions of the rabbit CE contain precursors and that the number of precursors is significantly higher in the peripheral CE than in the central region. The rate of secondary sphere formation was significantly higher for cells from the peripheral region than from the central region. Moreover, the progeny of rabbit CE spheres from the peripheral and central regions did not show any differences in mesenchymal and neuronal marker expression (TM, unpublished observation, 2004). These findings imply that rabbit CE precursors reside preferentially in the peripheral region of the CE and have potent self-renewal capacity compared with the central CE while demonstrating similar multipotentiality of precursors from both regions.

Neural crest cells, from which the CE originates,1 2 migrate and differentiate during corneal development in two waves.28 29 In the first step, the corneal epithelium forms and synthesizes the primary stroma—that is, periocular mesenchymal cells of neural crest origin—after which neural crest cells migrate to the margin of the optic cup and migrate between the lens and the corneal epithelium to contribute to development of the CE and trabecular meshwork. During the second wave of migration, neural crest cells invade the primary stroma and differentiate into corneal keratocytes. This embryological process may be compatible with our finding that more precursors with a high proliferative potential reside at the edge to the CE and supply differentiated cells centripetally to the central cornea during early development.

Rabbit CE precursors that are highly proliferative in vitro should proliferate in the anterior chamber of the eye because rabbit CE can proliferate at sites of CE defects in vivo. Under physiological conditions, CE are arrested in the G1-phase of the cell cycle,30 31 and TGF-ß2 in the aqueous humor suppresses entry into the S-phase.32 Cell–cell contact inhibition may also be an important mechanism for inducing cell cycle arrest to maintain the mature endothelial monolayer in a nonproliferative state.32 Although various transplantation techniques have been proposed for CE defects,20 33 34 35 36 37 the existence of CE precursors suggests that in vivo stimulation of precursors in the human CE by growth factors or other unknown chemical mediators may activate the proliferative potential of these precursors, leading to a novel treatment strategy for bullous keratopathy.

In rodents, the CE can repopulate after injury, but migration and enlargement without proliferation are the main mechanisms of wound healing for the human CE. Indeed, data based on assessment of the rabbit CE with proliferative capacity in vivo may not be relevant to the adult human CE from donor corneas. Further investigation is required to elucidate the distribution and proliferative capacity of precursors in the adult human CE. Our study, however, may contribute to the understanding of CE embryology, CE changes with aging, and the CE in the infant shortly after birth.

In summary, we compared the distribution and self-renewal capacity of rabbit CE precursors from the central and peripheral regions of the cornea by the sphere-forming assay. Our findings demonstrated that the peripheral and central CE contain a significant number of precursors and that the peripheral region of the rabbit CE has a higher density of precursors with stronger self-renewal capacity than the central region.


   Footnotes
 
Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and by a fund of Daiwa Securities Health Foundation.

Submitted for publication May 20, 2005; revised June 15, 2005; accepted August 23, 2005.

Disclosure: T. Mimura, None; S. Yamagami, None; S. Yokoo, None; M. Araie, None; S. Amano, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Satoru Yamagami, Department of Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, Hongo 7–3-1, Bunkyo-ku, Tokyo, Japan 113-8655; syamagami-tky{at}umin.ac.jp.


   References
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 Abstract
 Materials and Methods
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 Discussion
 References
 

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