**5. Treatment of bullous keratopathy with precursors derived from cultured spheres**

#### **5.1. Cryoinjury and injection of spheres into the anterior chamber**

Animals were handled in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. New Zealand White rabbits (weighing 2.0–2.4 kg, n=24) were anesthetized with an intramuscular injection of ketamine hydrochloride (60 mg/kg; Sankyo, Tokyo, Japan) and xylazine (10 mg/kg; Bayer, Leverkusen, Germany). To detach the CE from Descemet's membrane, a brass dowel cooled in liquid nitrogen was touched onto the cornea nine times (at the center and at eight peripheral sites). This procedure was repeated twice. Then the anterior chamber was washed three times with PBS through a 1.5-mm paracentesis.

To estimate the number of spheres needed to cover the inner surface of the cornea (Descemet's membrane), DiI-labeled spheres were seeded onto the denuded Descemet's membrane of freshly excised rabbit corneas and the mean area covered per sphere was found to be 1.2 ± 0.2 mm2 on day 7 (Fig. 5B). Therefore, it was calculated that 75 spheres were needed to cover a cornea. To allow for loss of spheres that failed to adhere, 150 DiI-labeled HCEC spheres or 1.0 × 107 HCECs were injected into the anterior chamber of the right eye after cryoinjury. Then the rabbits were maintained in the eyes-down position (Descemet's membrane down) for 24 hours to allow attachment (sphere eyes-down group, n=6). Cryoinjury alone (cryo group, n=6), injection of cultured HCECs with the eyes-down position being maintained for 24 hours (HCEC group, n=6), and injection of spheres in the eyes-up position (sphere eyes-up group, n=6) were also tested as controls. However, injection of cultured HCECs or injecting spheres in the eyes-up position did not reduce corneal edema in our preliminary study (Mimura T, unpublished observation, 2003), so these controls were not used in the present study. Each eye was inspected 2 or 3 times a week and was photographed on postoperative days 7, 14, 21, and 28. Central corneal thickness was measured with an ultrasonic pachymeter having a range of 0 to 1,200 μm (Tomey, Nagoya, Japan) and intraocular pressure was determined with a pneumatic tonometer (model 30 Classic; Mentor O & O, Norwell, MA) at 1, 3, 7, 14, 21, and 28 days after surgery. The average of three readings was obtained each time. One-way analysis of variance and Scheffe's multiple comparison test were used to compare mean values.

#### **5.2. Findings after surgery**

**Figure 5.** Migration of sphere-derived cells during culture for 7 days, modified from Mimura et al. (2005b) with per‐ mission. DiI-labeled spheres were seeded onto the denuded Descemet's membranes of rabbit corneas and cultured for 1 week in a humidified incubator. (A) Representative photographs of cell migration around an adherent DiI-la‐ beled sphere. Scale bar=100 μm. (B) Mean area occupied by cells migrating from the spheres on each day (n=10).

Changes of the potential difference (Fig. 4B) and short circuit current (Fig. 4C) were compared between human donor corneas without epithelium and HCEC-coated collagen sheets constructed with cells from spheres. The average potential difference and short circuit current of the HCEC-coated sheets ranged from 81% to 100% at 1, 5, and 10 minutes, corresponding to the results for normal human donor corneas denuded of epithelium. These findings suggested that the cultured HCEC spheres could generate CE-like cells with

min

**SHORT CIRCUIT CURRENT**

ouabain

**1 5 10 13 15**

**0**

Fig. 4. Morphology (A) and transport activity (B, C) of cells from cultured HCEC spheres, modified from Mimura et al. (2005b) with permission. (A) Confluent cells cultured in DMEM containing 10% FBS show the characteristic hexagonal shape of corneal endothelial cells. Changes of the potential difference (B) and short circuit current (C) for human donor corneas without epithelium and HCEC-coated collagen sheets (mean SD). The mean potential difference and short circuit current after 1, 5, and 10 minutes ranged from 81% to 100% for the HCEC-coated sheets, similar to the results for human corneas denuded of epithelium, indicating that the HCEC-like cells generated in culture had adequate transport

**Figure 4.** Morphology (A) and transport activity (B, C) of cells from cultured HCEC spheres, modified from Mimura et al. (2005b) with permission. (A) Confluent cells cultured in DMEM containing 10% FBS show the characteristic hexago‐ nal shape of corneal endothelial cells. Changes of the potential difference (B) and short circuit current (C) for human donor corneas without epithelium and HCEC-coated collagen sheets (mean ± SD). The mean potential difference and short circuit current after 1, 5, and 10 minutes ranged from 81% to 100% for the HCEC-coated sheets, similar to the results for human corneas denuded of epithelium, indicating that the HCEC-like cells generated in culture had ade‐ quate transport activity. When Na+ - K+ ATPase inhibitor ouabain was added to the chamber, the potential difference

decreased to 0 mV and the short circuit current declined to 0 μA in all cases.

**5**

**10**

**µA**

**C**

**15**

adequate transport activity.

440 Regenerative Medicine and Tissue Engineering

**A**

Our previous studies had suggested that cultured HCEC precursors have a limited selfrenewal capacity and mainly differentiate into HCEC-like cells. Then we investigated the use of precursors derived from cultured HCECs in a rabbit model of corneal endothelial damage. In the cryo and HCEC groups, the mean corneal thickness ranged from 953 ± 182 to 1,200 ± 0 μm (mean ± SD), as shown in Figure 6A. The mean (±SD) corneal thickness of the sphere eyesup group (704 ± 174 μm) was significantly less than that of the cryo group (1,011 ± 190 μm; P=0.006) and the HCEC group (953 ± 182 μm; P=0.022) after 28 days of observation, but the corneas were still edematous in the eyes-up group (Fig. 6A). In contrast, the corneal thickness decreased rapidly in the sphere eyes-down group, and the corneas were significantly thinner than in the other three groups after 14 (672 ± 90 μm), 21 (483 ± 84 μm), and 28 (394 ± 26 μm) days (*P*=0.006; Fig. 6A). Representative anterior segment photographs from the cryo group (Fig. 6B), HCEC group (Fig. 6C), and sphere eyes-up group (Fig. 6D ) show that the corneas of rabbits from these groups were edematous and displayed stromal opacity. In contrast, corneas from the sphere eyes-down group corneas became clear and the anterior chamber was easily visualized (Fig. 6E). No apparent inflammatory reactions suggesting rejection were observed by slit lamp microscopy throughout the postoperative period. On day 14, the intraocular pressure of the sphere eyes-up group was significantly higher than that of the cryo group (P=0.013). However, there was no increase of intraocular pressure (a possible side effect) on any other day in any group (Table 1).

Injection of spheres in the eyes-down position, but not injection of differentiated cultured HCECs or injection of spheres in the eyes-up position, restored endothelial function and decreased corneal edema in this rabbit model of bullous keratopathy model. These findings suggest that injection of spheres derived from cultured HCECs and maintenance of an eyesdown position for 24 hours may be a potential treatment strategy for corneal endothelial defects that is less invasive compared with conventional full-thickness corneal transplantation.

**Figure 6.** Changes of corneal thickness and other findings in a rabbit model of bullous keratopathy, modified from Mimura et al. (2005b) with permission. (A) Mean corneal thickness decreases gradually in the sphere eyes-down group (closed circles, n=6). It is significantly less than in the cryo group (open circles, n=6), HCEC group (closed triangles, n=6), and sphere eyes-up group (open triangles, n=6) on days 14, 21, and 28 (\*P<0.001 by one-way analysis of variance and Scheffe's multiple comparison test). (B–E) Representative photographs of corneas from each group. The cornea is opa‐ que in the cryo group (B), HCEC group (C), and sphere eyes-up group (D), and the anterior chamber is not well visual‐ ized. In contrast, there is no corneal opacity in the sphere eyes-down group (E).


**Table 1.** Intraocular pressure in each group after surgery (mm Hg). Data represent the mean ± SD for six rabbits. \**P*=0.013 by one-way analysis of variance and Scheffe's multiple comparison test.

#### **5.3. Histologic findings**

than in the other three groups after 14 (672 ± 90 μm), 21 (483 ± 84 μm), and 28 (394 ± 26 μm) days (*P*=0.006; Fig. 6A). Representative anterior segment photographs from the cryo group (Fig. 6B), HCEC group (Fig. 6C), and sphere eyes-up group (Fig. 6D ) show that the corneas of rabbits from these groups were edematous and displayed stromal opacity. In contrast, corneas from the sphere eyes-down group corneas became clear and the anterior chamber was easily visualized (Fig. 6E). No apparent inflammatory reactions suggesting rejection were observed by slit lamp microscopy throughout the postoperative period. On day 14, the intraocular pressure of the sphere eyes-up group was significantly higher than that of the cryo group (P=0.013). However, there was no increase of intraocular pressure (a possible side effect) on

Injection of spheres in the eyes-down position, but not injection of differentiated cultured HCECs or injection of spheres in the eyes-up position, restored endothelial function and decreased corneal edema in this rabbit model of bullous keratopathy model. These findings suggest that injection of spheres derived from cultured HCECs and maintenance of an eyesdown position for 24 hours may be a potential treatment strategy for corneal endothelial defects that is less invasive compared with conventional full-thickness corneal transplantation.

**preop 1 3 7 14 21 28**

**Figure 6.** Changes of corneal thickness and other findings in a rabbit model of bullous keratopathy, modified from Mimura et al. (2005b) with permission. (A) Mean corneal thickness decreases gradually in the sphere eyes-down group (closed circles, n=6). It is significantly less than in the cryo group (open circles, n=6), HCEC group (closed triangles, n=6), and sphere eyes-up group (open triangles, n=6) on days 14, 21, and 28 (\*P<0.001 by one-way analysis of variance and Scheffe's multiple comparison test). (B–E) Representative photographs of corneas from each group. The cornea is opa‐ que in the cryo group (B), HCEC group (C), and sphere eyes-up group (D), and the anterior chamber is not well visual‐

**Time (days)**

\*

\* \*

Cryo HCE Sphere eyes-up Sphere eyes-down

any other day in any group (Table 1).

442 Regenerative Medicine and Tissue Engineering

ized. In contrast, there is no corneal opacity in the sphere eyes-down group (E).

**(μm)**

**A**

**Corneal thickness**

Examination of hematoxylin & eosin-stained sections revealed that corneas from the cryo group (Fig. 7A), HCEC group (Fig. 7B), and sphere eyes-up group (Fig. 7C) were thickened and no cells could be detected on Descemet's membrane. In contrast, a monolayer of cells had formed on Descemet's membrane in the sphere eyes-down group, and there was no edema and no mononuclear cell infiltration of the posterior stroma (Fig. 7D). In the cryo group (Figs. 8A, 8E), HCEC group (Figs. 8B, 8F), and sphere eyes-up group (Figs. 8C, 8G), no HCECs (Figs. 8A–C) with positive staining for DiI (Figs. 8E–G) were found on Descemet's membrane at the central cornea in flat mount preparations. In contrast, HCEC-like hexagonal cells were detected at this site in the sphere eyes-down group (Fig. 8D). These cells were DiI-positive (Fig. 8H), indicating that they had originated from the injected spheres and not from the host. In the sphere eyes-down group, DiI-negative cells were present in the peripheral cornea, but all cells in the central and paracentral (8 mm in diameter) cornea were DiI-positive. The density of HCECs in the six grafts of the sphere eyes-down group at 28 days after surgery ranged from 2,625 to 2,875 cells/mm2 , with a mean (±SD) value of 2,781 ± 92 cells/mm2 . Before surgery, the density of endothelial cells in the rabbit cornea was from 3,300 to 3,500 cells/mm2 . In the sphere eyes-down group, very few DiI-positive cells were detected in the inferior trabecular mesh‐ work or on the iris, whereas a number of DiI-positive cells were attached at these sites in the HCEC group and the sphere eyes-up groups (data not shown).

Cells adherent to the inner surface of the cornea (Descemet's membrane) were DiI-positive in the sphere eyes-down group, indicating that these were HCECs derived from the injected spheres and not residual host cells. In addition, DiI-positive cells were rarely detected in the trabecular meshwork or on the surface of the iris, so the spheres mainly attached to and spread over the cornea in the eyes-down group. These results suggested that sphere-derived HCECs could restore corneal hydration after sphere transplantation.

**Figure 7.** Histologic findings, modified from Mimura et al. (2005b) with permission. In the cryo group (A), HCEC group (B), and sphere eyes-up group (C), stromal edema is prominent and no cells are present on Descemet's membrane at the central cornea. In contrast, a monolayer of cells can be detected on Descemet's membrane in the sphere eyesdown group (D). There is no mononuclear cell infiltration near Descemet's membrane, suggesting no rejection of the xenogeneic cells (D). Scale bar=100 μm, hematoxylin & eosin stain.

**Figure 8.** Flat mount preparations with phase-contrast (A–D) and fluorescence (E–H) microscopy, modified from Mim‐ ura et al. (2005b) with permission. At the central cornea, there are no cells on Descemet's membrane in the cryo group (A, E), HCEC group (B, F), and sphere eyes-up group (C, G). In contrast, HCEC-like hexagonal cells are present in the sphere eyes-down group (D). These cells are also DiI-positive (H). Scale bar=100 μm.

#### **5.4. Advantages of transplanting CE precursors**

For regenerative medicine, amplification of stem cells is required to treat each tissue or organ. Although much attention has been paid to maintaining the undifferentiated nature ("stemness") of stem cells and promoting their amplification, the molecular mechanisms of stem cell replication and differentiation are still not fully understood. In comparison with amplification of adult stem cells, cultured cells can be used more easily to produce tissuecommitted precursors by the sphere-forming assay, as demonstrated in our studies. Similar techniques to produce abundant precursors should be tested for various tissues as a method of obtaining cells for regenerative medicine.

Transplantation of HCEC precursors into the anterior chamber has several advantages over penetrating keratoplasty with a full-thickness donor cornea. For example, complications associated with open-sky surgery (expulsive hemorrhage and the risk of wound dehis‐ cence) are essentially eliminated. In addition, several postoperative complications, such as irregular astigmatism, wound leakage, corneal infection, neovascularization, and persis‐ tent epithelial defects, can be avoided when using the combined approach. After conven‐ tional full-thickness human corneal allografting with local and/or systemic immunosuppressants, the leading cause of failure is graft rejection (Price et al., 1991; Wilson & Kaufman, 1990). Although there was no apparent inflammatory reaction histologically, we cannot deny the possibility of allograft rejection over the long term because nonadher‐ ent cells should migrate out of the anterior chamber. It is noteworthy that injection of HCEC precursors did not improve bullous keratopathy created by scraping off endothe‐ lial cells in rabbits (data not shown). This may be because cryoinjury to the cornea, but not endothelial cell scraping, promoted the proliferation and migration of HCEC s that led to recovery of corneal clarity.
