Intraocular pressure in each group after surgery (mm Hg). Data represent the mean ± SD for six rabbits. *
The cornea is composed of a multilayered epithelium, Bowman's membrane, stroma, Descemet's membrane, and endothelium. The corneal endothelium (CE) is a single layer of hexagonal cells that separates the corneal stroma from the aqueous humor of the anterior chamber. Transparency of the cornea is maintained by regulation of stromal hydration through the barrier and pump functions of the CE, and corneal transplantation has long been used to treat corneal endothelial defects. In fact, more than half of the patients who undergo full-thickness corneal transplantation have impairment of visual acuity due to corneal endothelial problems alone and have a normal corneal epithelium (Cosar et al., 2002; Mannis et al., 1981; Rapuano et al., 1990). Corneal transplantation requires a fresh human cornea, but there is a worldwide shortage of donors (Barboza et al., 2007; Cao et al., 2006; Shimazaki et al., 2004; Tuppin et al., 2007).
Stem cells or progenitor cells are defined by a capacity for self-renewal and the ability to generate different types of cells (multipotentiality) that are involved in the formation of mature tissues. In contrast, precursor cells are unipotential cells with limited proliferative capacity. Regenerative stem cells or precursors can be detected by the sphere-forming assay in various adult tissues, including the central nervous system (Nunes et al., 2003), bone marrow (Krause et al., 2001), skin (Kawase et al., 2004; Toma et al., 2001), retina (Coles et al., 2004), corneal epithelium (Mimura et al., 2010a; Yokoo et al., 2008), corneal stroma (Amano et al., 2006; Mimura, 2008a, 2008b; Uchida et al., 2005; Yamagami et al., 2007), and corneal endothelium (Amano et al., 2006; Mimura, 2005a, 2005b, 2005c, 2007, 2010b; Yamagami, 2006, 2007; Yokoo et al., 2005).
Despite the successful isolation and characterization of stem cells from various tissues, relatively few animal studies have been done to investigate the efficacy of stem cell transplantation. A three-dimensional carrier that maintains cell-to-cell interactions is indispensable for tissue engineering using stem cells, but the resulting structural complexity does not allow us to easily perform investigations of stem cell transplantation.
We have isolated precursors with the propensity to develop into corneal endothelial-like cells from the CE of human donor corneas (Yokoo et al., 2005). We have also demonstrated that cultured human corneal endothelial cells (HCECs) and rabbit CE-derived precursors are an effective cell source for treating corneal endothelial defects in a rabbit model (Mimura 2005a, 2005b). Because the number of corneal endothelial cell (CEC) precursors that can be isolated from a native cornea is insufficient for corneal transplantation, establishment of a method for the mass production of precursor cells is required before CEC transplantation can be employed clinically.
In this chapter, we introduce our recent work in the fields of regenerative medicine and tissue engineering for the CE using bipotential precursor cells. We isolated precursors with the propensity to develop into CECs from human CE, and we investigated the distribution and proliferative capacity of precursor cells derived from the central and peripheral regions of the cornea by the sphere-forming assay. We also tested the effect of injecting human corneal endothelial spheres anterior chamber (instead of full-thickness corneal transplantation) in a rabbit model of bullous keratopathy, a condition associated with corneal endothelial defects.
2. Origin and development of the Corneal Endothelium
Neural crest cells, from which the CE is derived (Bahn et al., 1984; Johnston et al., 1979), migrate and differentiate in two waves during corneal development (Liu et al., 1998; Meier et al., 1982). In the first wave, the corneal epithelium is formed by periocular mesenchymal cells of neural crest origin and it synthesizes the primary stroma, after which neural crest cells migrate to the margin of the optic cup and then migrate between the lens and corneal epithelium to contribute to development of the CE and the trabecular meshwork. In the second wave, neural crest cells invade the primary stroma and differentiate into corneal keratocytes.
3. Isolation of sphere colonies from human Corneal Endothelium
3.1. Primary sphere-forming assay
This study was conducted in accordance with the Declaration of Helsinki. Corneas were obtained from the Central Florida Lions Eye Tissue Bank and the Rocky Mountain Lions’ Eye Bank at 4 to 10 days after death. The age of the donors was 41 to 78 years. The CE and Descemet’s membrane were peeled away in a sheet from the periphery to the center of the inner surface of the cornea with fine forceps, as described previously (Sakai et al., 2002). To avoid the inclusion of posterior stromal tissue, we only used endothelium that was smoothly peeled off together with Descemet’s membrane. The harvested CE was incubated at 37ºC for 3 hours in basal medium containing 0.02% collagenase (Sigma-Aldrich, St. Louis, MO). This was followed by incubation in 0.2% ethylenediaminetetraacetic acid (EDTA) at 37ºC for 5 minutes, and then dissociation into single cells by trituration with a fire-polished Pasteur pipette. The viability of the isolated CECs was >90%, as shown by trypan blue staining (Wako Pure Chemical Industries, Osaka, Japan). After addition of a trypsin inhibitor (Invitrogen-Gibco), the cells were resuspended in basal medium and the number of cells was counted (Coulter counter; Beckman-Coulter, Hialeah, FL). Neither cytokeratin-3 nor cytokeratin-12 expression was detected, indicating that the cells thus obtained were all CECs without contamination by other corneal cell types.
Half of the cells were labeled with a fluorescent cell tracker (CM-DiI; C-7000; Molecular Probes, Eugene, OR), as described elsewhere (Mimura et al. 2004), to examine sphere formation by reaggregation. DiI-labeled cells and unlabeled cells were mixed and seeded at a density of 1 cell/μL (250 cells/cm2), 10 cells/μL (2,500 cells/cm2), 30 cells/μL (7,500 cells/cm2), or 50 cells/μL (12,500 cells/cm2) on 60-mm uncoated dishes containing 5 mL of medium for floating culture (Reynolds & Weiss, 1992, 1996) (Fig. 1B). No spheres were generated in the cultures with only 1 viable cell/μL, but numerous spheres were formed at 30 and 50 cells/μL, with some arising from reaggregation as indicated by DiI staining. Spheres were completely DiI-positive or DiI-negative when culture was performed at 10 cells/μL (Fig. 1C), indicating that these spheres were derived from proliferation and not from reaggregation of the dissociated cells.
Incubation was done in a humidified incubator under an atmosphere of 5% CO2, with 40 ng/mL basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF) being added to the medium every other day. To investigate whether the isolated cells were contaminated with corneal epithelial cells, expression of epithelial markers such as keratins K3 and K12 (Irvine et al., 1997; Moll et al., 1982) was assessed by the reverse transcription–polymerase chain reaction (RT-PCR) before the start of culture. Then primary culture was performed and the existence of fibroblast-like cells was investigated to assess contamination by stromal cells. CECs were isolated without contamination by corneal epithelial cells, as demonstrated by RT-PCR analysis of corneal epithelial markers (K3 and K12 genes), as well as the characteristic hexagonal shape of the cells in primary culture (data not shown). Almost complete disaggregation into single cells was achieved, since counting of single, double, and triple cells showed that 99% of all cells were single (Fig. 1B).
After incubation for 5 days, small floating spheres formed. These spheres grew larger after 10 days, while the nonproliferating cells died and were eliminated (Fig. 1D). After 10 days, we only counted cell clusters with a diameter of at least 50 μm, in order to distinguish growing spheres from dying ones. To verify that the increase of colony size was actually due to cell proliferation, we added the thymidine analogue BrdU to cultures at 24 hours before fixation. Then the spheres were stained with an FITC-conjugated anti-BrdU antibody (1:100; Roche Diagnostics, Basel, Switzerland) at room temperature (RT) for 60 minutes in the dark. We found that BrdU labeled most of the cells in each sphere on day 10 (Fig. 1E), indicating that the spheres contained proliferating cells. These results suggested that the sphere colonies arose from single isolated HCECs and that the sphere-forming cells possess the capacity to proliferate. When the number of spheres obtained was counted after 10 days of culture, we found that 257 ± 83 spheres (mean ± SD, n=8) were generated per dish (50,000 cells). In a typical case, 2.5 ± 104 cells were isolated from a 10-mm piece of corneal tissue, generating approximately 130 spheres after 10 days. These spheres had a diameter of 88.3 ± 15.9 μm (mean ± SD, n=35). The replating efficiency showed a dramatic decline between primary and secondary sphere colonies. When the primary spheres were trypsinized and incubated in serum-free floating culture, secondary colonies were generated (Fig. 1F) at a level of approximately 15 ± 1 (n=3) per dish of 10,000 cells. This suggests that HCECs have the capacity for self-renewal and formation of sphere colonies, but this capacity is limited.
3.2. Distribution of sphere colonies derived from human Corneal Endothelial cells
HCECs were obtained from the central cornea (up to 7.5 mm from the center) and the peripheral cornea (from 7.5 to 10 mm) (Fig. 1A). As a result, the number of primary sphere colonies per 5,000 cells (mean ± SD) was significantly higher when peripheral HCECs were used (13.6 ± 3.5 spheres/5,000 cells) than when central HCECs were used (3.3 ± 1.6 spheres/5,000 cells) (Fig. 1G). The rate of sphere formation by HCECs from the peripheral cornea was approximately 4 times that for HCECs from the central cornea in repeated experiments (data not shown).
It has generally been accepted that human CE does not proliferate after birth, but our findings and some previous reports suggest that the CE may undergo slow proliferation
3.3. Characterization of primary spheres derived from human Corneal Endothelium
Immunocytochemical analysis of 10-day spheres was performed as follows. The spheres were fixed with methanol (Wako Pure Chemical Industries) in phosphate-buffered saline (PBS) for 10 minutes, washed in PBS, and incubated for 30 minutes with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X20 (BSA/PBST) to block nonspecific staining. Then, the spheres were incubated for 2 hours at RT with the following specific primary antibodies diluted in BSA/PBST: mouse anti-vimentin monoclonal antibody (mAb) (1:300; Dako, Glostrup, Denmark), mouse anti-nestin mAb (1:200; BD PharMingen, San Diego, CA), rabbit anti-p75 neurotrophin receptor (p75 NTR) polyclonal antibody (pAb) (1:200; Promega Corp., Tokyo, Japan), mouse anti-neurofilament 145 mAb (NFM, 1:400; Chemicon, Temecula, CA), rabbit anti 3-tublin pAb (1:2000; Covance Research Products, Denver, PA), rabbit anti-glial fibrillary acidic protein (GFAP) pAb (1:400; Dako), mouse anti-O4 mAb (1:10; Chemicon), rabbit anti-peripherin pAb (1:100; Chemicon), and mouse anti-α-smooth muscle actin (α-SMA) mAb (1:200; Sigma-Aldrich). As a control, mouse IgG (1:1000; Sigma-Aldrich) or normal rabbit serum (1:1000; Dako) was used instead of the primary antibody. After the spheres were washed in PBS, incubation was done for 1 hour at RT with the appropriate secondary antibody diluted in BSA/PBST. The secondary antibodies were fluorescent-labeled goat anti-mouse IgG (Alexa Fluor 488, 1:200; Molecular Probes) and fluorescent-labeled goat anti-rabbit IgG (Alexa Fluor 594, 1:400; Molecular Probes). Nuclei were counterstained with Hoechst 33342 (1:2000; Molecular Probes). After another wash in PBS, the spheres were examined under a laser scanning confocal microscope (Fluoview; Olympus, Tokyo, Japan). When anti-O4 or anti-p75NTR mAb was used, the permeabilization step was omitted.
Figure 2A shows a bright-field image of a typical sphere colony. Spheres derived from HCECs were not stained by nonimmune mouse IgG (Fig. 2D) or normal rabbit serum (Fig. 2G). Nestin has been used as a marker for the detection of immature neural progenitor cells in multipotential sphere colonies derived from the brain (Gage, 2000), skin (Toma et al., 2001), inner ear (Li et al., 2003), retina (Tropepe et al., 2000), corneal epithelium (Mimura et al., 2010a; Yokoo et al., 2008), corneal stroma (Amano et al., 2006; Mimura 2008a, 2008b; Uchida et al., 2005; Yamagami et al., 2007), and CE (Amano et al., 2006; Mimura 2005a, 2005b, 2005c, 2007, 2010b; Yokoo et al., 2005, Yamagami, 2006, 2007). Expression of α-SMA (a marker of mesenchymal myofibroblasts) and expression of p75 NTR (a marker of neural crest stem cells) was also investigated by immunocytochemistry because HCECs are derived from the neural crest. Cells in the spheres showed immunoreactivity for nestin (Fig. 2B) and for α-SMA (Fig. 2C), but not for p75 NTR (data not shown). Next, the spheres were immunostained for various neural markers. As a result, spheres were found to be positive for an immature neuronal marker (β3-tubulin, Fig. 2E) and an astroglial marker (GFAP, Fig. 2F), but not a mature neuronal marker (NFM), an oligodendroglial marker (O4), or a peripheral nerve neuronal marker (peripherin; data not shown). These findings indicated that spheres isolated from human donor CE contain bipotential precursors that are capable of undergoing differentiation into mesenchymal cells and neuronal cells.
3.4. Secondary sphere formation
To further evaluate the proliferative capacity of HCECs, cells from the primary spheres were passaged under the same conditions as those used for the initial sphere culture. On day 10, primary spheres were treated with 0.05% trypsin/0.02% EDTA and dissociated into single cells, which were added to 24-well culture plates at a density of 10 cells/μL in medium containing primary culture supernatant. These cells were then incubated for a further 10 days in basal medium.
Secondary spheres were generated from the dissociated primary spheres, but the yield of secondary sphere colonies was lower than after primary culture. Although self-renewal potential was indicated by the ability of cells from individual primary spheres to form secondary spheres, this potential was limited, as evidenced by the failure of sphere formation at the third passage. These results indicated that the precursor cells had a limited proliferative capacity. Photographs of representative secondary spheres are shown in Figure 1F.
3.5. Differentiation of sphere colonies
Individual primary spheres (day 10) were transferred to 13 mm glass coverslips coated with 50 μg/ml poly-L-lysine (PLL) and 10 μg/ml fibronectin (BD Biosciences, Billerica, MA) in separate wells, as described previously (Reynolds & Weiss, 1992). To promote differentiation, 1% fatal bovine serum (FBS) was added to the basal medium, and culture was continued for another 7 days. Immunocytochemical examination of spheres and their progeny was performed after 7 days of adherent culture on glass coverslips.
To investigate whether sphere progeny possessed the characteristics of mesenchymal or neural cells, single spheres (day 10) were transferred onto PLL/laminin-coated glass coverslips in medium containing 1% or 15% FBS or onto bovine ECM-coated culture plates in medium containing 15% FBS. Spheres remained adherent to the PLL/laminin-coated glass coverslips, but cells migrated out from the spheres grown on glass coverslips coated with bovine ECM alone. After 7 days, some of the cells that had migrated from the spheres showed double immunostaining for nestin and β3-tublin (Fig. 2H), as has been reported for human scalp tag–derived cells (Toma et al., 2001). However, there was no staining of cells migrating out of the spheres for α-SMA, p75NTR, NFM, peripherin, GFAP, or O4.
RT-PCR was performed to examine the expression of genes governing the proteins detected by immunocytochemistry in the spheres and their progeny (Fig. 2I). GAPDH mRNA was detected in both spheres and progeny, but not in the control assay without the RT reaction. Expression of nestin,β 3-tublin, GFAP, and α-SMA mRNA was detected in the spheres and adherent progeny after 35 PCR cycles. However, mRNAs for NFM, p75NTR, and peripherin were not found under any cycling conditions. Nestin andβ 3-tublin mRNAs were also detected in HCECs from primary culture.
These findings indicated that spheres isolated from human CE contain bipotential precursors, yielding progeny that display the morphologic characteristics of HCECs. Taken together, these results suggest that precursors from the CE remain close to the tissue of origin and undergo differentiation into CECs. Because precursors should ideally differentiate efficiently to produce their tissue of origin, precursors obtained from the CE may be more appropriate for tissue regeneration or cell transplantation than those derived from the multipotential stem cells.
4. Isolation of precursors from cultured human Corneal Endothelial cells
4.1. Culture of human Corneal Endothelial cells
As mentioned in sections 3.1-3.4, we have isolated precursor cells from human donor corneas (Yamagami et al. 2007; Yokoo et al., 2005). However, the number of precursors that can be isolated from a cornea is insufficient for corneal endothelial regeneration, so establishment of a mass production method for precursor cells is needed before clinical application can be attempted. Accordingly, we isolated spheres from cultured HCECs and investigated whether the cells of these spheres had CE-like functions. We also tested the effect of injecting these spheres into the anterior chamber (instead of full-thickness corneal transplantation) in a rabbit model of bullous keratopathy, representing a state in which corneal endothelial defects exist.
Several groups have established HCEC culture techniques (Chen et al., 2001; Engelmann & Friedl 1989; Miyata et al., 2001; Yue et al. 1989). Various growth factors have been reported to influence the proliferation of cells cultured from human CE, including fibroblast growth factor (Chen et al., 2001; Engelmann 1988, 1989, 1995; Yue et al. 1989; Samples et al., 1991), epidermal growth factor (Chen et al., 2001; Samples et al., 1991; Schultz et al., 1992; Yue et al. 1989), nerve growth factor (Chen et al., 2001), and endothelial cell growth supplement (Blake et al., 1997; Yue et al. 1989). In addition, cell attachment and growth can be supported by seeding cells onto an artificial matrix, such as chondroitin sulfate or laminin (Engelmann et al., 1988), laminin-5 (Yamaguchi et al., 2011), extracellular matrix secreted by bovine corneal endothelial cells (Blake et al., 1997; Miyata et al., 2001), or fibronectin/type I collagen coating mix (Joyce & Zhu, 2004).
In our studies, HCECs were isolated and cultured according to the published protocols of Joyce and our laboratory with some modifications (Chen et al., 2001; Joyce & Zhu, 2004; Miyata et al., 2001). Briefly, Descemet’s membrane was carefully dissected with the intact CE. After centrifugation, membrane strips were incubated in 0.02% EDTA solution at 37ºC for 1 hour to loosen intercellular junctions. Then isolated cells were plated in 6-well tissue culture plates that had been precoated with undiluted fibronectin/type I collagen coating mix, and incubation was done at 37ºC under a humidified atmosphere with 5% CO2. After primary cultures reached confluence, cells were subcultured at a 1:4 ratio, and cells from the 4th to 6th passages were used.
4.2. Isolation and characterization of sphere colonies
Cells from the 4th or 5th passages were used in this study. HCECs were incubated in 0.2% EDTA at 37ºC for 5 minutes and then were dissociated into single cells by pipetting with a flame-polished Pasteur pipette. The viability of the isolated HCECs was >90% as shown by trypan blue staining. The sphere-forming assay was used for primary culture (Reynolds & Weiss, 1992). Cells were plated at a density of 10 viable cells/μL (40,000 cells per well or 1,420 cells/cm2) in the uncoated wells of 60-mm culture dishes. The basal medium was Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with B27, epidermal growth factor (EGF, 20 ng/mL), and basic fibroblast growth factor (bFGF, 20 ng/mL). A methylcellulose gel matrix (1.5%; Wako) was added to the medium to prevent reaggregation of the cells (Gritti et al., 1999; Kawase et al., 2004). To distinguish growing spheres from dying cell clusters, only spheres with a diameter of more than 50 μm were counted. For passaging, primary spheres were harvested on day 7 and treated with 0.5% EDTA for dissociation into single cells, which were plated in 24-well culture plates at a density of 10 cells/μL. Then culture was continued for another 7 days in basal medium containing the methylcellulose gel matrix.
Spheres formed after 7 days of culture (Fig. 3A), while nonproliferating cells were eliminated. Many of the cells in each sphere were BrdU-positive (Fig. 3B), indicating that such cells were proliferating. These findings suggested that the spheres had developed from single HCECs and that the sphere-forming cells displayed proliferative activity. The number of sphere colonies obtained after 7 days of culture was 44 ± 10 per 10,000 cells (mean ± SD). Replating of primary spheres to generate secondary sphere colonies was less efficient, indicating that the cells only had limited self-renewal capacity.
On immunostaining, the spheres were positive for nestin (Fig. 3C), which is a marker of immature cells (Lendahl et al., 1990), and for α-SMA (Fig. 3D), a mesenchymal myofibroblast marker. We previously demonstrated that primary spheres derived from human donor CE express β-III tubulin and GFAP, a mature glial cell marker, as well as nestin and α-SMA (Fig. 2), but β-III tubulin and GFAP were negative in the spheres derived from cultured HCECs.
4.3. Differentiation of sphere colonies
Individual primary spheres (day 7) were transferred to 13-mm glass coverslips coated with 50 μg/mL PLL and 10 μg/mL fibronectin in separate wells (Mimura et al., 2005a). To promote differentiation, 1% or 15% FBS was added to the basal medium, after which culture was continued for another 7 days.
Then the spheres were transferred to PLL/fibronectin-coated glass coverslips in 24-well plates and were cultured in a differentiation medium containing 1% or 15% fetal bovine serum (FBS). After 7 days, many cells were found to have migrated out of the spheres. Fewer than 5% of these cells were α-SMA-positive (Fig. 3E), whether cultured with 1% or 15% FBS. All of these cells were negative for control IgG (Fig. 3F) and for the differentiated epithelial cell marker cytokeratin 3, as well as for nestin, β-III tubulin, and GFAP (not shown). These findings indicated that a single sphere colony could give rise to a small population of mesenchymal cells under clonogenic conditions. Expression of nestin and α-SMA by the spheres, as well as α-SMA expression by their progeny, was confirmed using RT-PCR (Fig. 3G). Positivity for β-III tubulin mRNA was only detected in cultures with 1% FBS.
Spheres derived from donor CE expressed an immature cell marker (nestin), an immature neuronal marker (β-III tubulin), and a mature glial cell marker (GFAP), while their progeny expressedβ -III tubulin and nestin, but not GFAP. In contrast, the spheres and progeny obtained from cultured HCECs did not express neuronal markers and showed decreased expression of immature cell markers. These findings suggested that the precursors were close in nature to the original tissue and underwent differentiation during culture. Thus, precursors obtained from cultured HCECs may be a more appropriate cell source than cells from donor CE, because precursors that efficiently differentiate into the tissue of origin are ideal for tissue regeneration or cell transplantation.
4.4. Assessing the pump function of cells derived from spheres
The pump function of four collagen sheets seeded with cells derived from HCEC spheres was measured in an Ussing chamber, as reported previously with some modifications (Wigham, 1981, 2000; Hodson & Wigham 1983). The collagen sheets were obtained from the Nippi Biomatrix Research Institute (Tokyo, Japan). Cells from HCEC spheres were suspended at 5.0 × 106 cells in 1.5 mL of culture medium and transferred to circular collagen sheets (10 mm in diameter). Each sheet was placed in one well of a 24-well plate, and the plate was centrifuged at 1,000 rpm (176 g) for 10 minutes to enhance cell attachment. Then the sheets were incubated in culture medium for 2 days, after which nonadherent cells and debris were removed (Fig. 4A). Human donor corneas with the epithelium removed mechanically (n=4), plain collagen sheets (n=4), or HCEC-coated collagen sheets (n=4) were mounted in the Ussing chamber.
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 adequate transport activity.
4.5. Migration and proliferation of spheres on rabbit descemet’s membrane
Animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primary HCEC spheres (cultured for 7 days) were labeled with a fluorescent cell tracker (CM-DiI). After the endothelium was gently scraped off four freshly excised rabbit corneas with a sterile cotton swab, HCEC spheres were applied to the posterior surface of each cornea. Then the corneas were placed in 24-well plates and maintained in culture medium for 7 days. HCECs that migrated onto the corneas were detected under a fluorescence microscope, and the area occupied by fluorescent cells migrating from the spheres was measured with the NIH image program (n=10).
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
Our previous studies had suggested that cultured HCEC precursors have a limited self-renewal 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 eyes-up 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 (
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 eyes-down 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.
|Cryo||12.7 ± 1.9||10.8 ± 1.8||10.5 ± 4.3*||11.0 ± 1.6||10.2 ± 1.5|
|HCEC||11.8 ± 1.5||11.6 ± 2.8||12.3 ± 4.5||11.8 ± 2.2||11.5 ± 2.8|
|Sphere (eyes-up)||13.2 ± 2.0||9.9 ± 1.9||17.2 ± 4.0*||13.3 ± 3.4||10.5 ± 3.1|
|Sphere (eyes-down)||13.5 ± 3.2||8.7 ± 2.1||14.8 ± 2.2||13.7 ± 1.7||11.7 ± 1.9|
5.3. Histologic findings
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 meshwork 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.
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 tissue-committed 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 dehiscence) are essentially eliminated. In addition, several postoperative complications, such as irregular astigmatism, wound leakage, corneal infection, neovascularization, and persistent epithelial defects, can be avoided when using the combined approach. After conventional 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 nonadherent 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 endothelial 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.
We demonstrated that the endothelium from the peripheral region of the human cornea contains a higher density of precursors with strong proliferative capacity compared to the central endothelium. These HCEC precursors are able to differentiate into both mesenchymal and neural cells. We have also established a method for mass production by isolation of precursors from cultured HCECs using the sphere-forming assay. Transplantation of spheres into the anterior chamber and short-term maintenance of the eyes-down position was shown to be a simple and effective treatment strategy in our rabbit model of bullous keratopathy. This method of managing corneal endothelial defects may have the potential to replace conventional full-thickness corneal grafting and compensate for the worldwide shortage of donor corneas.
AcknowledgmentsThis work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Amann J Holley G. P Lee S. B Edelhjunauser H. F 2003Increased endothelial cell density in the paracentral and peripheral regions of the human cornea. Am J Ophthalmol, 135 5May 2003), 584 590 0002-9394
Amano S Yamagami S Mimura T Uchida S Yokoo S 2006Corneal stromal and endothelial cell precursors. Cornea, 25 10Suppl 1), (December 2006), S73 S77 0277-3740
Bahn C. F Falls H. F Varley G. A Meyer R. F Edelhauser H. F Bourne W. M 1984Classification of corneal endothelial disorders based on neural crest origin. Ophthalmology, 91 6June 1984), 558 563 0161-6420
Barboza A. P Pereira R. C Garcia C. D Garcia V. D 2007Project of cornea donation in the hospital complex of santa casa de porto alegre, brazil. Transplant Proc, 39 2March 2007), 341 343 0041-1345
Blake D. A Yu H Young D. L Caldwell D. R 1997Matrix stimulates the proliferation of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci, 38 6May 1997), 1119 1129 0146-0404
Boisjoly H. M Tourigny R Bazin R Laughrea P. A Dube I Chamberland G Bernier J Roy R 1993Risk factors of corneal graft failure. Ophthalmology, 100 11November 1993), 1728 1735 0161-6420
Cao K. Y Dorrepaal S. J Seamone C Slomovic A. R 2006Demographics of corneal transplantation in canada in 2004. Can J Ophthalmol, 41 6December 2006), 688 692 0008-4182
Chen K. H Azar D Joyce N. C 2001Transplantation of adult human corneal endothelium ex vivo: A morphologic study. Cornea, 20 7October 2001), 731 737 0277-3740
Coles B. L Angenieux B Inoue T Del Rio-Tsonis, K., Spence, J. R., McInnes, R. R., Arsenijevic, Y., & van der Kooy, D. ( 2004Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci U S A, 101 44November 2004), 15772 15777 1091-6490
Cosar C. B Sridhar M. S Cohen E. J Held E. LAlvim Pde, T., Rapuano, C. J., Raber, I. M., & Laibson, P. R. ( 2002Indications for penetrating keratoplasty and associated procedures, 1996-2000. Cornea, 21 2March 2002), 148 151 0277-3740
Engelmann K Friedl P 1989Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol, 25 11November 1989), 1065 1072 0883-8364
Engelmann K Friedl P 1995Growth of human corneal endothelial cells in a serum-reduced medium. Cornea, 14 1January 1995), 62 70 0277-3740
Engelmann K Bohnke M Friedl P 1988Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci, 29 11November 1988), 1656 1662 0146-0404
Gage F. H 2000Mammalian neural stem cells. Science, 287 5457February 2000), 1433 1438 0036-8075
Gritti A Frolichsthal-schoeller P Galli R Parati E. A Cova L Pagano S. F Bjornson C. R Vescovi A. L 1999Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci. 19 9May 1999), 3287 3297 1529-2401
Hodson S Wigham C 1983The permeability of rabbit and human corneal endothelium. J Physiol, 342September 1983), 409 419 0022-3751
Irvine A. D Corden L. D Swensson O Swensson B Moore J. E Frazer D. G Smith F. J Knowlton R. G Christophers E Rochels R Uitto J Mclean W. H 1997Mutations in cornea-specific keratin k3 or k12 genes cause meesmann’s corneal dystrophy. Nat Genet, 16 2June 1997), 184 187 1061-4036
Johnston M. C Noden D. M Hazelton R. D Coulombre J. L Coulombre A. J 1979Origins of avian ocular and periocular tissues. Exp Eye Res, 29 1July 1979), 27 43 0014-4835
Joyce N. C Zhu C. C 2004Human corneal endothelial cell proliferation: Potential for use in regenerative medicine. Cornea, 23 8Suppl, (November 2004), S8 S19 0277-3740
Kanai A Tanaka M Ishii R Nakajima A 1982Bullous keratopathy after anterior-posterior radial keratotomy for myopia for myopic astigmatism. Am J Ophthalmol, 93 5May 1982), 600 606 0002-9394
Kawano H Uesugi Y Nakayasu K Kanai A 2003Long-term follow-up for bullous keratopathy after sato-type anterior-posterior corneal refractive surgery. Am J Ophthalmol, 136 6December 2003), 1154 1155 0002-9394
Kawase Y Yanagi Y Takato T Fujimoto M Okochi H 2004Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth. Exp Cell Res, 295 1April 2004), 194 203 0014-4827
Krause D. S Theise N. D Collector M. I Henegariu O Hwang S Gardner R Neutzel S Sharkis S. J 2001Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell, 105 3May 2001), 369 377 0092-8674
Lendahl U Zimmerman L. B Mckay R. D 1990Cns stem cells express a new class of intermediate filament protein. Cell, 60 4February 23 1990), 585 595 0092-8674
Li H Liu H Heller S 2003Pluripotent stem cells from the adult mouse inner ear. Nat Med, 9 10October 2003), 1293 1299 1078-8956
Liu C. Y Shiraishi A Kao C. W Converse R. L Funderburgh J. L Corpuz L. M Conrad G. W Kao W. W 1998The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development. J Biol Chem, 273 35August 1998), 22584 22588 0021-9258
Mannis M. J Krachmer J. H 1981Keratoplasty: A historical perspective. Surv Ophthalmol, 25 5March-April 1981), 333 338 0039-6257
Meier S 1982The distribution of cranial neural crest cells during ocular morphogenesis. Prog Clin Biol Res, 82 1 15 0361-7742
Mimura T Yamagami S Yokoo S Usui T Tanaka K Hattori S Irie S Miyata K Araie M Amano S 2004Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci, 45 9September 2004), 2992 2997 0146-0404
Mimura T Yamagami S Yokoo S Yanagi Y Usui T Ono K Araie M Amano S 2005aSphere therapy for corneal endothelium deficiency in a rabbit model. Invest Ophthalmol Vis Sci, 46 9September 2005), 3128 3135 0146-0404
Mimura T Yokoo S Araie M Amano S Yamagami S 2005bTreatment of rabbit bullous keratopathy with precursors derived from cultured human corneal endothelium. Invest Ophthalmol Vis Sci, 46 10October 2005), 3637 3644 0146-0404
Mimura T Yamagami S Yokoo S Araie M Amano S 2005cComparison of rabbit corneal endothelial cell precursors in the central and peripheral cornea. Invest Ophthalmol Vis Sci, 46 10October 2005), 3645 3648 0146-0404
Mimura T Yamagami S Usui TSeiichi, Honda, N., & Amano, S. ( 2007Necessary prone position time for human corneal endothelial precursor transplantation in a rabbit endothelial deficiency model. Curr Eye Res, 32 7-8July-August 2007), 617 623 1460-2202
and distribution of rabbit keratocyte precursors. Mimura T Amano S Yokoo S Uchida S Usui T o Yamagami S 2008aIsolati n Mol Vis, 14January 2008), 197 203 1090-0535
Mimura T Amano S Yokoo S Uchida S Yamagami S Usui T Kimura Y Tabata Y 2008bTissue engineering of corneal stroma with rabbit fibroblast precursors and gelatin hydrogels. Mol Vis, 14January 2008), 1819 1828 1090-0535
Mimura T Yamagami S Uchida S Yokoo S Ono K Usui T Amano S 2010aIsolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions. Mol Vis, 16August 2010), 1712 1719 1090-0535
Mimura T Yamagami S Yokoo S Usui T Amano S 2010bSelective isolation of young cells from human corneal endothelium by the sphere-forming assay. Tissue Eng Part C Methods, 16 4August 2010), 803 812 1937-3384
Miyata K Drake J Osakabe Y Hosokawa Y Hwang D Soya K Oshika T Amano S 2001Effect of donor age on morphologic variation of cultured human corneal endothelial cells. Cornea, 20 1January 2001), 59 63 0277-3740
Moll R Franke W. W Schiller D. L Geiger B Krepler R 1982The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell, 31 1November 1982), 11 24 0092-8674
Nunes M. C Roy N. S Keyoung H. M Goodman R. R Mckhann G 2nd, Jiang, L., Kang, J., Nedergaard, M., & Goldman, S. A. ( 2003Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med, 9 4April 2003), 439 447 1078-8956
Price F. W Jr., Whitson, W. E., & Marks, R. G. ( 1991Graft survival in four common groups of patients undergoing penetrating keratoplasty. Ophthalmology, 98 3March 1991), 322 328 0161-6420
Rapuano C. J Cohen E. J Brady S. E Arentsen J. J Laibson P. R 1990Indications for and outcomes of repeat penetrating keratoplasty. Am J Ophthalmol, 109 6June 15 1990), 689 695 0002-9394
Reynolds B. A Weiss S 1992Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 255 5052March 1992), 1707 1710 0036-8075
Reynolds B. A Weiss S 1996Clonal and population analyses demonstrate that an egf-responsive mammalian embryonic cns precursor is a stem cell. Dev Biol, 175 1Apr 10 1996), 1 13 0012-1606
Sakai R Kinouchi T Kawamoto S Dana M. R Hamamoto T Tsuru T Okubo K Yamagami S 2002Construction of human corneal endothelial cdna library and identification of novel active genes. Invest Ophthalmol Vis Sci, 43 6June 2002), 1749 1756 0146-0404
Samples J. R Binder P. S Nayak S. K 1991Propagation of human corneal endothelium in vitro effect of growth factors. Exp Eye Res, 52 2Feb 1991), 121 128 0014-4835
Schultz G Cipolla L Whitehouse A Eiferman R Woost P Jumblatt M 1992Growth factors and corneal endothelial cells: Iii. Stimulation of adult human corneal endothelial cell mitosis in vitro by defined mitogenic agents. Cornea, 11 1Jan 1992), 20 27 0277-3740
Shimazaki J Shinozaki N Shimmura S Holland E. J Tsubota K 2004Efficacy and safety of international donor sharing: A single-center, case-controlled study on corneal transplantation. Transplantation, 78 2Jul 27 2004), 216 220 0041-1337
Toma J. G Akhavan M Fernandes K. J Barnabe-heider F Sadikot A Kaplan D. R Miller F. D 2001Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol, 3 9September 2001), 778 784 1465-7392
Tropepe V Coles B. L Chiasson B. J Horsford D. J Elia A. J Mcinnes R. R Van Der Kooy D 2000Retinal stem cells in the adult mammalian eye. Science, 287 5460March 2000), 2032 2036 0036-8075
Tuppin P Esperou H Delbosc B Loty B 2007corneal graft activity in france (1990-2005): Decreasing the gap between supply and demand]. J Fr Ophtalmol, 30 5May 2007), 475 482 0181-5512
Uchida S Yokoo S Yanagi Y Usui T Yokota C Mimura T Araie M Yamagami S Amano S 2005Sphere formation and expression of neural proteins by human corneal stromal cells in vitro. Invest Ophthalmol Vis Sci, 46 5May 2005), 1620 1625 0146-0404
Wigham C Hodson S 1981The effect of bicarbonate ion concentration on trans-endothelial short circuit current in ox corneas. Curr Eye Res, 1 1 37 41 0271-3683
Wigham C. G Turner H. C Swan J Hodson S. A 2000Modulation of corneal endothelial hydration control mechanisms by rolipram. Pflugers Arch, 440 6Oct 2000), 866 870 0031-6768
Williams K. A Roder D Esterman A Muehlberg S. M Coster D. J 1992Factors predictive of corneal graft survival. Report from the australian corneal graft registry. Ophthalmology, 99 3Mar 1992), 403 414 0161-6420
Wilson S. E Kaufman H. E 1990Graft failure after penetrating keratoplasty. Surv Ophthalmol, 34 5Mar-Apr 1990), 325 356 0039-6257
Yamagami S Suzuki Y Tsuru T 1996Risk factors for graft failure in penetrating keratoplasty. Acta Ophthalmol Scand, 74 6Dec 1996), 584 588 1395-3907
Yamagami S Mimura T Yokoo S Takato T Amano S 2006Isolation of human corneal endothelial cell precursors and construction of cell sheets by precursors. Cornea, 25 10Suppl 1), (December 2006), S90 S92 0277-3740
Yamagami S Yokoo S Mimura T Takato T Araie M Amano S 2007Distribution of precursors in human corneal stromal cells and endothelial cells. Ophthalmology, 114 3March 2007), 433 439 0161-6420
Yamaguchi M Ebihara N Shima N Kimoto M Funaki T Yokoo S Murakami A Yamagami S 2011Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5. Invest Ophthalmol Vis Sci, 52 2Feb 2011), 679 684 0146-0404
Yokoo S Yamagami S Usui T Amano S Araie M 2008Human corneal epithelial equivalents for ocular surface reconstruction in a complete serum-free culture system without unknown factors. Invest Ophthalmol Vis Sci, 49 6June 2008), 2438 2443 0146-0404
Yokoo S Yamagami S Yanagi Y Uchida S Mimura T Usui T Amano S 2005Human corneal endothelial cell precursors isolated by sphere-forming assay. Invest Ophthalmol Vis Sci, 46 5May 2005), 1626 1631 0146-0404
Yue B. Y Sugar J Gilboy J. E Elvart J. L 1989Growth of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci, 30 2Feb 1989), 248 253 0146-0404