Amniotic Membrane as a Carrier for Cultivated Human
`Corneal Endothelial Cell Transplantation
`
`Yutaka Ishino,1 Yoichiro Sano,1 Takahiro Nakamura,1 Che J. Connon,1 Helen Rigby,2
`Nigel J. Fullwood,2 and Shigeru Kinoshita1
`
`PURPOSE. It would be advantageous if cultivated human corneal
`endothelial cells (cHCECs) could be transplanted for the treat-
`ment of diseases caused by corneal endothelial disorders. To
`achieve this, a matrix that can serve as a carrier for cHCECs is
`needed. The present study was conducted to examine the
`feasibility of using amniotic membrane (AM) as a carrier for this
`application.
`METHODS. HCECs obtained from peripheral corneal tissue were
`cultivated, passaged, and transplanted onto denuded AM. The
`cell density and morphology of the resultant cHCECs on AM
`were examined by light, scanning electron, and transmission
`electron microscopy. To determine whether these cHCEC
`sheets on AM carrier were functional in vivo, the cHCEC sheets
`on AM were transplanted onto rabbit corneas whose Descem-
`et’s membrane and endothelial cells had been completely re-
`moved. After transplantation, the corneal appearance was ex-
`amined by slit lamp biomicroscopy, and corneal thickness was
`measured daily by pachymetry. At 7 days after surgery, the
`grafts were examined by light, scanning electron, and trans-
`mission electron microscopy.
`RESULTS. The density of the cHCECs on AM was greater than
`3000 cells/mm2. Morphologically, the cHCEC sheets consisted
`of a fairly continuous layer of flat squamous polygonal endo-
`thelial cells that appeared uniform in size with tightly opposed
`cell junctions in vitro and in vivo after transplantation. The
`corneas that received transplanted cHCEC sheets had little
`edema and retained their thinness and transparency.
`CONCLUSIONS. The cell density and morphology of cHCECs on
`AM were similar to those of normal corneas, and cHCECs on
`AM were functional in vivo. These results indicate that AM
`maintains HCEC morphology and function and could serve as a
`carrier for cHCEC transplantation. (Invest Ophthalmol Vis Sci.
`2004;45:800 – 806) DOI:10.1167/iovs.03-0016
`
`C orneal endothelium is essential for the maintenance of
`
`normal corneal hydration, thickness, and transparency.1
`Corneal endothelium of human has essentially no regenerative
`capacity in vivo.2,3 Serious corneal endothelial cell loss causes
`irreversible corneal edema. The type of corneal transplantation
`
`From the 1Department of Ophthalmology, Kyoto Prefectural Uni-
`versity of Medicine, Kyoto, Japan; and 2Biological Sciences, Lancaster
`University, Lancaster, United Kingdom.
`Supported by Research Grants 13557145 and 14770970 from the
`Japanese Ministry of Education, Culture and Science, the Wellcome
`Trust, and the T. F. C. Frost Trust.
`Submitted for publication January 7, 2003; revised June 24 and
`August 28, 2003; accepted August 31, 2003.
`Disclosure: Y. Ishino, None; Y. Sano, None; T. Nakamura,
`None; C.J. Connon, None; H. Rigby, None; N.J. Fullwood, None; S.
`Kinoshita, None
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be marked “advertise-
`ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`Corresponding author: Yutaka Ishino, Department of Ophthalmol-
`ogy, Kyoto Prefectural University of Medicine, 465 Hirokoji Kawara-
`machi, Kamigyo-ku, Kyoto, Japan; yishino@ophth.kpu-m.ac.jp.
`
`800
`
`layers are damaged.
`performed depends on which corneal
`Penetrating keratoplasty is generally the treatment of choice
`for eyes with a damaged endothelial layer such as in Fuchs’
`endothelial dystrophy and iatrogenic bullous keratopathy.
`However, there are several concerns with penetrating kerato-
`plasty: rejection; suture problems, which can cause astigma-
`tism and infection; and denervation, which affects corneal
`function. In addition, in many countries, including Japan, the
`supply of donor corneas is insufficient. Therefore, the ideal
`situation would be if only corneal endothelial cells could be
`transplanted to treat corneal endothelial diseases. Further-
`more, it would be highly desirable if small numbers of corneal
`endothelial cells could be expanded in culture, and these
`cultivated human corneal endothelial cells (cHCECs) could be
`transplanted into eyes of many patients.
`For cHCEC transplantation in vivo, some type of carrier is
`obviously necessary. Up to now, for corneal endothelial cell
`transplantation, gelatin membranes (Schwartz BD, et al. IOVS
`1980;21:ARVO Abstract 100; McCully JP, et al. IOVS 1981;22:
`ARVO Abstract 230)4,5 and coated hydrogel lenses6 have been
`used as synthetic carriers for these cells. Human amniotic
`membrane (AM), widely used as a surgical material,9 has been
`used successfully as a carrier for cultivated corneal epithelial
`cell transplantation.8 –10 In this study, we sought to examine
`whether AM could also serve as a carrier for cHCECs, both in
`vitro and in vivo.
`
`MATERIALS AND METHODS
`
`Human Corneal Tissue
`
`A donor cornea (47 years old, male, endothelial cell density: 3065
`cells/mm2) obtained from Northwest Lions Eye Bank was preserved
`(Optisol GS; Chiron Vision, Irvine, CA) and transported to our hospital
`on ice. After using its center for human corneal transplantation the
`residual limbal tissue was used for this study at day 7 after death.
`
`HCEC Culture
`
`To cultivate HCECs, corneal limbal tissue was placed in a Petri dish
`containing Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen
`Corp., Carlsbad, CA), 50 U/mL penicillin, and 50 ␮g/mL streptomycin.
`Under a dissecting microscope, Descemet’s membrane with its at-
`tached corneal endothelium 1 mm apart from trabecular meshwork
`was stripped from the stroma and placed in a 35-mm dish containing
`1.2 U/mL dispase in phosphate-buffered saline (PBS). The tissue was
`incubated for 1 hour at 37°C, and the cells were rinsed gently with a
`sterile pipette. The dispase was then inactivated by suspending the
`cells in a medium containing DMEM, 50 U/mL penicillin, and 50 ␮g/mL
`streptomycin. After gentle centrifugation (3 minutes at 180g), the cells
`were resuspended in culture medium containing DMEM, 50 U/mL
`penicillin, 50 ␮g/mL streptomycin, 10% fetal bovine serum (ICN Bio-
`medicals, Inc., Aurora, OH), and 2 ng/mL basic fibroblast growth factor
`(Invitrogen Corp.).
`The cells were incubated in wells of a collagen IV– coated 24-well
`plate at 37°C in 5% carbon dioxide-95% humidified air. The medium
`was changed every other day. Cells reached confluence in 10 to 20
`
`Investigative Ophthalmology & Visual Science, March 2004, Vol. 45, No. 3
`Copyright © Association for Research in Vision and Ophthalmology
`
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`IOVS, March 2004, Vol. 45, No. 3
`
`Amniotic Membrane as a Carrier for cHCEC Transplants
`
`801
`
`days and were then subcultured by treatment with trypsin-EDTA (In-
`vitrogen Corp.) and seeded at a ratio of 1:2 to 1:8.
`
`Preparation of AM
`In accordance with the tenets of the Declaration of Helsinki and with
`proper informed consent, human AMs were obtained at the time of
`cesarean section. The method of removing the amniotic epithelial cells
`from the AM has been reported.11 Briefly, human AM was stored at
`⫺80°C in DMEM and glycerol (Nacalai Tesque, Kyoto, Japan) after the
`AM was washed with PBS containing antibiotics (5 mL of 0.3% ofloxa-
`cin). Immediately before use, the thawed AM was deprived of amniotic
`epithelial cells by incubation with 0.02% EDTA (Wako Pure Chemical
`Industries, Osaka, Japan) at 37°C for 2 hours, followed by gentle cell
`scraping with a cell scraper (Nalge Nunc International, Naperville, IL).
`The tissues were then washed twice with sterile PBS. To confirm
`whether epithelium was completely removed from AM, light micros-
`copy was used to examine the AM (Fig. 1A).
`
`Seeding cHCECs on Denuded AM
`Denuded AMs were spread, basement membrane side up, on the
`bottom of polyester culture inserts (Corning Corp., Corning, NY), and
`the inserts were placed in wells of a 12-well plate. Confluent mono-
`layers of cHCECs from passage 5 were trypsinized, centrifuged, and
`resuspended at a final cell-seeding concentration of 6.0 ⫻ 103 cells/
`mm2 (1.2 ⫻ 106 cells/mL ⫻ 0.5 mL of cell suspension per 100 mm2 of
`culture area). Resuspended cells were gently seeded on denuded AM
`spread on culture inserts in wells of a 12-well plate, centrifuged gently
`(3 minutes at 180g) to increase the cell density of the cHCEC sheets
`and to increase uniform attachment to the denuded AM, and incubated
`at 37°C in 5% carbon dioxide-95% humidified air. The culture medium
`was changed 3 days later and then every other day for 2 weeks.
`In some experiments, to investigate the extent of the survival of
`cHCECs on AM after transplantation in vivo, we labeled the cHCECs
`with the fluorescent membrane dye DiI (1,1-dioctadecyl-3,3,3,3-tetra-
`methylindocarbocyanine perchlorate).12 The stock solution of DiI was
`made by dissolving 5 mg dye in 5 mL 70% ethanol. The DiI labeling
`solution was made by diluting the stock solution by 10. Immediately
`before seeding the HCECs on to the AM, the cells were labeled in vitro
`by adding DiI solution to the cell suspension. After incubation for 5
`minutes at 37°C, the cell suspension was cooled on ice for 15 minutes.
`Excess dye was then removed by washing the cells twice in PBS. Cells
`were resuspended at a final cell-seeding concentration of 6.0 ⫻ 103
`cells/mm2 and seeded on denuded AM, cultured for 7 days, and trans-
`planted. We conducted this experiment with two cHCEC sheets on AM
`carriers transplanted into rabbit corneas. At 7 days after transplanta-
`tion, we killed the animals, removed the grafts, and observed them
`under the fluorescence microscope. We stained same tissues with
`alizarin red and examined them under the microscope.
`
`Light and Electron Microscopy
`Cultures of cHCECs on denuded AM were examined by light, scanning
`electron, and transmission electron microscopy.
`For light microscopy, tissues were stained with alizarin red and
`hematoxylin and eosin. For alizarin red staining, day-14 cultures on AM
`were placed endothelial side up on glass slides. Tissues were briefly
`rinsed in 0.9% sodium chloride followed by a 1-minute staining with 1%
`alizarin red in deionized water. Cell density (cells per square millime-
`ter) was calculated by averaging cell density of three cHCEC sheets.
`For cell density of one sheet, five areas (each area equaled 0.25 mm2)
`per one sheet were examined and averaged.
`For hematoxylin and eosin stain, day-14 cultures on AM were
`embedded in optimal cutting temperature (OCT) compound (Tissue-
`Tek; Sakura Fine Technical, Tokyo, Japan). After freezing, the resultant
`blocks were cut into 8-␮m-thick sections with a cryostat. The sections
`were stained with hematoxylin and eosin for examination.
`For scanning electron microscopy, day 14 cultures on AM were
`fixed in 2.5% glutaraldehyde in 0.1 M PBS, washed three times for 15
`
`(A) Denuded AM before cHCECs were seeded. No amniotic
`FIGURE 1.
`epithelial cells were observed. (B) Light micrograph of cHCECs culti-
`vated on denuded AM for 2 weeks. Flatmount stained with alizarin red.
`The cHCECs appeared as a fairly continuous monolayer with a cell
`density of 3340 cells/mm2. (C) Cross section of the cHCECs on de-
`nuded AM on a culture insert (CI). A continuous intact monolayer of
`cHCECs is evident (arrow). Magnification: (A) ⫻100; (C) ⫻200.
`
`minutes in PBS, postfixed for 2 hours in 2% osmium tetroxide, and
`washed three more times in PBS. After dehydration through a graded
`ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) specimens were
`transferred to hexamethyldisilazane (Agar Scientific, London, UK) for
`2 ⫻ 10 minutes and allowed to air dry. When dry, specimens were
`mounted on aluminum stubs and sputter coated with gold before
`examination on a scanning electron microscope (model JSM 5600;
`Japanese Electron Optical Limited [JEOL], Tokyo, Japan).
`For transmission electron microscopy, day-14 cultures on AM were
`fixed in 2.5% glutaraldehyde in 0.1 M PBS, postfixed in 2% osmium
`tetroxide, dehydrated through a graded ethanol series, and embedded
`in epoxy resin (Agar 100; Agar Scientific). Ultrathin (70 nm) sections
`
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`

`IOVS, March 2004, Vol. 45, No. 3
`
`HCECs cultivated on AM revealed a continuous layer of flat,
`squamous, polygonal endothelial cells that appeared uniform
`in size. The interdigitations at the cell boundaries were not as
`distinct as those in normal endothelium. These cells had a few
`pits and vacuoles, but appeared to be healthy, well developed,
`and closely attached to one another, with tightly opposed cell
`junctions (Fig. 2). Transmission electron microscopic images
`showed a monolayer of flat endothelial cells, healthy and well
`formed, with tightly opposed cell junctions. Adjoining cells
`overlapped each other slightly to maintain maximum contact,
`as would be expected. At the base of the cHCEC basement
`membrane, material was clearly being produced (Fig. 3).
`
`Evaluation of Corneal Appearance and Thickness
`after Transplantation
`At 4 days after the operation, the control grafts, consisting of
`only stripped Descemet’s membrane, became highly edema-
`tous (Fig. 4A) as did the control grafts with only acellular AM
`(Fig. 4B). However, grafts with cHCECs on AM had little edema
`and excellent transparency, and we could see the transplanted
`cHCEC sheets clearly through the transparent corneal button
`(Fig. 4C). No signs of rejection or neovascularization were
`observed. This situation continued for at least 7 days after
`transplantation (Fig. 5). Corneal grafts with cHCECs were sig-
`
`802
`
`Ishino et al.
`
`TABLE 1. Transplantation Groups
`
`Group
`
`Stripping DM
`
`Transplantation
`
`HCEC
`SD Control
`AM Control
`TO Control
`
`(⫹)
`(⫹)
`(⫹)
`(⫺)
`
`cHCEC sheet
`(⫺)
`Acellular AM only
`(⫺)
`
`were collected on copper grids and stained for 1 hour each with uranyl
`acetate and 1% phosphotungstic acid and for 20 minutes with Reynolds
`lead citrate before examination on a transmission electron microscope
`(JEM 1010; JEOL).
`
`Animals
`Male Japanese white rabbits weighing 2 to 3 kg were obtained from
`Shimizu Laboratory (Kyoto, Japan). All animals were treated in accor-
`dance with the ARVO Statement for Use of Animals in Ophthalmic and
`Vision Research. The Committee for Animal Research, Kyoto Prefec-
`tural University of Medicine, approved all animal studies.
`
`cHCEC Transplantation
`Transplantation was performed on the right eye only. Corneal buttons
`and graft beds were prepared by excising a 7.00-mm site in the central
`cornea. Descemet’s membranes together with the endothelium were
`stripped from the corneal buttons. cHCEC sheets, using AM as a
`carrier, on culture inserts were trephined to a diameter of 6.25 mm,
`and they were separated gently from inserts with fine forceps. These
`sheets were placed on the stroma of the corneal buttons, left for a few
`minutes until dry to secure the sheet to the stroma. The corneal
`buttons with cHCEC sheets were then placed on the graft bed of the
`same animal and sutured with eight interrupted sutures and a contin-
`uous suture (10-0 nylon). Four groups were prepared in this experi-
`ment (Table 1). Each group had three animals. In the first group (the
`cHCEC group), after Descemet’s membrane was stripped, the cHCEC
`sheet was placed on the corneal button and transplanted as just
`described. In the second group (the SD control group), Descemet’s
`membrane was stripped and the button transplanted as described. In
`the third group (the AM control group), after Descemet’s membrane
`was stripped, just the acellular AM was transplanted, as described. In
`the last group (the TO control group), the host cornea was trephined
`only and transplanted as described, without stripping Descemet’s
`membrane and transplanting any sheet. All grafted eyes were examined
`every day after transplantation. Grafts with technical difficulties (e.g.,
`hyphema, infection, or loss of the anterior chamber) were excluded
`from further consideration. At day 4 after transplantation, the inter-
`rupted sutures were removed.
`
`Evaluation of Corneal Appearance, Thickness,
`and Histology after Transplantation
`Each day after transplantation, corneal appearance was examined by
`slit lamp biomicroscopy, and corneal thickness was determined with
`an ultrasonic pachymeter (model SP-2000; Tomey, Nagoya, Japan).
`Corneal thickness is a measure of corneal endothelial function.13 The
`mean of 10 measured values was calculated. At 7 days after transplan-
`tation, we killed the cHCEC sheet transplant recipients, removed the
`grafts, and observed them by light and electron microscopes.
`
`RESULTS
`
`Light and Electron Microscopy of the cHCECs
`Sheet In Vitro
`The density of cHCECs seeded on AM was 3285.3 ⫾ 62.0
`cells/mm2, the cHCECs appeared as a fairly continuous mono-
`layer (Figs. 1B, 1C). Scanning electron microscopic images of
`
`FIGURE 2.
`Scanning electron micrograph of cultured HCECs. The cells
`were cultivated on denuded AM for 2 weeks. (A) Cells formed a
`continuous monolayer layer on the AM. (B) At higher magnification, it
`was evident that the cells were polygonal, fairly uniform in size, and in
`close contact with each other with tightly opposed cell junctions.
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`IOVS, March 2004, Vol. 45, No. 3
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`Amniotic Membrane as a Carrier for cHCEC Transplants
`
`803
`
`FIGURE 4. At 4 days after the operation, the control corneas consist-
`ing of stripped Descemet’s membrane (A) and acellular AM (B) became
`highly edematous. The grafts consisting of HCECs on AM (C) had little
`edema and excellent transparency.
`
`Assessment of Cultured HCECs Transplanted
`In Vivo
`Before transplantation, cHCECs were present on AM at high
`density (Fig. 8A). One week after transplantation, the cells
`were still present on the AM carrier and maintained a high
`density (2410.0 ⫾ 31.1 cells/mm2; Fig. 8B). In addition, no
`proliferation or migration of cHCECs was observed beyond the
`edge of the AM (Fig. 8C). Alizarin red stain of the cHCEC sheet
`with corneal button showed that there were no endothelial
`cells between the cHCEC sheet and the host– graft junction.
`Therefore, rabbit corneal endothelial cells did not seem to have
`migrated into the corneal button at the time examined (Fig.
`8D).
`
`DISCUSSION
`
`We made cHCEC sheets using denuded acellular AM as a
`nonsynthetic carrier and transplanted them in vivo in our
`
`FIGURE 5. At 7 days after the operation, the control corneas consist-
`ing of stripped Descemet’s membrane (A) and acellular AM (B) were
`highly edematous. The grafts consisting of HCECs on AM (C) had little
`edema and corneal transparency.
`
`FIGURE 3. Transmission electron micrographs of cultivated HCECs
`cultivated on denuded AM for 2 weeks. (A) A continuous monolayer of
`these cells covered the surface of denuded AM. These cells had a few
`pits and vacuoles, but they kept normal endothelial features. (B)
`Adjoining cells either overlapped each other slightly or abutted to
`maintain good contact. Thick arrow: cell– cell junction; thin arrows:
`cells producing basement membrane material.
`
`nificantly thinner when compared with both the stripped De-
`scemet’s membrane and AM control groups in daily pachym-
`etry measurements (P ⬍ 0.05). The average value for rabbit
`corneal thickness before the operations was 350.7 ⫾ 10.2 ␮m
`(mean ⫾ SD). After the operation, average values for corneal
`thickness were always greater than 800 ␮m in the acellular AM
`and stripped Descemet’s control groups. However,
`in the
`HCEC group, the average values for corneal thickness was
`consistently below 500 ␮m from days 5 to 7 after the operation
`(day 5: 460.7 ⫾ 100.6 ␮m; day 6: 436.0 ⫾ 100.5 ␮m; day 7:
`460.7 ⫾ 117.7 ␮m, mean ⫾ SD). The average counts in the
`HCEC group were not significantly different from those of the
`TO group (Fig. 6).
`Light and electron microscopy of the HCEC sheet 7 days
`after transplantation showed a continuous monolayer of
`HCECs on AM. The cells were polygonal, fairly uniform in size,
`and in close contact with one another (Figs. 7A, 7B). A trans-
`mission electron microscopy image of the HCECs shows that a
`continuous monolayer of cHCECs covered the surface of the
`denuded AM. Apparent complexity of cell– cell junctions and
`cell–AM substrates were observed. Many mitochondria and
`lysosomes were present in the cytoplasm of cHCECs on AM
`(Fig. 7C).
`
`

`

`804
`
`Ishino et al.
`
`IOVS, March 2004, Vol. 45, No. 3
`
`FIGURE 6. Average corneal
`thick-
`ness (mean ⫾ SD) after transplanta-
`tion. The average corneal thickness
`was always greater than 800 ␮m in
`both the stripped Descemet’s mem-
`brane and the acellular AM control
`groups. In the cHCEC transplant re-
`cipients, the average corneal thick-
`ness was consistently less than that
`in both the control and AM groups.
`Between the TO control and cHCEC
`groups, there was no significant dif-
`ference in the average corneal thick-
`ness.
`
`present study. Cultivated HCEC transplantation on Descemet’s
`membrane in vitro has been reported,14 –16 and the cells were
`reported to form a stable monolayer and keep their character-
`istic construction. We think AM is one of the most feasible
`candidates for cHCEC transplantation, because AM can be
`obtained more easily than any other nonsynthetic carriers such
`as Descemet’s membrane, and it has been used for cultured
`corneal epithelial cell transplantation with good clinical re-
`sults. There have been several studies4,8,17–19 of corneal endo-
`thelial cell transplantation in vivo, but most of these do not
`involve corneal endothelial cells from humans, but instead
`cultured corneal endothelial cells from animals: rabbit, bovine,
`and cat. To our knowledge, in one study,19 human neonatal
`corneal endothelial cells were cultivated and seeded onto the
`Descemet’s membrane of the corneal button and then the cells
`
`transplanted into African green monkeys. Therefore, this is the
`first report of cHCECs transplanted in vivo using adult cHCECs.
`In our study, the morphology and structure of cHCECs
`transplanted on denuded AM were evaluated by vital staining,
`as well as scanning and transmission electron microscopy. We
`found that the ultrastructure and density of these cells was very
`similar to that of normal corneal endothelial cells ex vivo. It is
`also important in cHCEC transplantation to obtain higher cell
`density. Clinically, it has been proposed that donor corneal
`tissue with endothelial density of more than 2500 cells/mm2
`are ideal to be transplanted in patients with bullous kerato-
`plasty. Using centrifugation, we obtained cHCEC sheets with a
`density of more than 3000 cells/mm2. These cHCEC sheets
`seem to have high enough cell density to be used in patients
`for bullous keratopathy.
`
`FIGURE 7.
`Light and electron micro-
`graphs of HCECs 7 days after trans-
`plantation. (A) A fairly continuous
`monolayer of HCECs. (B) Scanning
`electron micrograph also showing
`that HCECs formed a continuous
`monolayer layer on the AM. They
`were polygonal,
`fairly uniform in
`size, and in close contact with one
`another. (C) A transmission electron
`micrograph of HCECs showing that a
`continuous monolayer of cHCECs
`covered the surface of denuded AM.
`Thin arrow: designate cell– cell junc-
`tion. Gray arrows: close association
`of basal plasma membrane and AM;
`thick arrows: mitochondria in cyto-
`plasm.
`
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`IOVS, March 2004, Vol. 45, No. 3
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`Amniotic Membrane as a Carrier for cHCEC Transplants
`
`805
`
`retained their corneal thickness and transparency. We think
`that the gap had some influence on the pump–leak balance.
`The water permeating into the corneal stroma through the
`nonendothelial area may pump out through the adjacent hu-
`man and rabbit endothelial area. Because an area of central
`cornea more than 6 mm in diameter is covered by transplanted
`HCECs,
`it is reasonable to speculate that endothelial cells
`pump water from corneal stroma, although other possible
`factors such as evaporation may contribute to this to some
`extent.
`In our present study, we transplanted cHCEC sheets by
`trephining the central corneas, removing Descemet’s mem-
`branes with corneal endothelial cells, placing cHCEC sheets on
`the stroma of corneal buttons, and suturing them. A technique
`termed posterior lamellar keratoplasty, an operation for the
`treatment of bullous keratopathy, has been reported by Melles
`et al.20 In this method the full-thickness cornea is not trans-
`planted,
`just the posterior lamella of the cornea, and the
`method could be adapted for cHCEC transplantation. We have
`now investigated a cHCEC transplantation technique to re-
`move corneal endothelial cells with Descemet’s membrane and
`transplant a cHCEC sheet through a corneoscleral incision,
`similar to posterior lamellar keratoplasty. cHCEC sheet trans-
`plantation by this technique would be expected to have the
`same advantages as a posterior lamellar keratoplasty: fewer
`problems with sutures after they are in place, lower astigma-
`tism, and more efficient use of donor tissue. In addition cHCEC
`transplantation may well have the advantage that scheduled
`operations could be performed, because we would not be
`dependent on the availability of corneoscleral discs. This pos-
`sibility therefore has many advantages for both patients and
`health professionals.
`cHCEC transplantation has the potential to be performed,
`not only as an allogeneic transplantation procedure but also as
`an autotransplantation procedure, if a small number of corneal
`endothelial cells from a healthy eye were cultivated, expanded,
`and transplanted to the contralateral endothelial damaged eye
`of the same patient. Moreover, in regenerative medicine, the
`potential of some pluripotent stem cells21–23 for use in clinical
`treatments has been noted. Therefore, if pluripotent stem cells
`(e.g., hematopoietic stem cells and mesenchymal stem cells
`obtained from bone marrow) could be obtained from patients
`who undergo bullous keratoplasty and these stem cells could
`be induced to differentiate into corneal endothelial cells, it
`would be possible to transplant autologous corneal endothelial
`sheets without any risk of rejection.
`
`References
`
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`present. Am J Obstet Gynecol. 1979;134:833– 845.
`8. Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S. Culti-
`vated corneal epithelial transplantation for ocular surface recon-
`
`FIGURE 8. DiI-labeled cHCECs on AM. (A) At day 0, before transplan-
`tation in vivo, the cHCECs were present on the AM at a high density.
`(B) At day 7 after transplantation, the cultured HCECs were present on
`AM. (C) At day 7 after transplantation, the cHCECs showed no evi-
`dence of proliferation or migration beyond the edge of the AM. (D)
`Between cHCEC sheet and host– graft junction in the Descemet-
`stripped area, there were no endothelial cells. Neither donor nor host
`endothelial cells migrated from the original region. (C, D) Solid line:
`edge of cHCEC sheet; dotted line: host– graft junction. Magnification:
`(A, B) ⫻100; (C, D) ⫻40.
`
`In our in vivo study, we also found that the rabbit corneas
`with transplanted cHCECs on AM retained their thickness and
`transparency compared with the controls for 1 week, and we
`also found, but in only one rabbit, that this situation continued
`for 4 weeks (data not shown). Because rabbit corneal endothe-
`lial cells have been known to proliferate in vivo, we wished to
`investigate the extent of survival of cHCECs transplanted in
`vivo by using DiI labeling. The results of the DiI labeling
`showed that the cHCECs remained on the denuded AM trans-
`planted onto the corneal button at least 4 weeks. These results
`show that transplanted cHCEC sheets remained and were func-
`tional for at least 4 weeks. We intend to further investigate the
`long-term consequences of cHCEC transplantation and the
`functions of the cHCEC sheet. The HCEC density in the trans-
`plants showed 27% reduction 7 days after transplantation,
`either because of the tissue damage at the time of surgery or
`the short life of some cultivated endothelial cells. To investi-
`gate the long-term consequences of cHCEC transplantation, it
`is not suitable to use rabbits as recipients, because their cor-
`neal endothelial cells proliferate in vivo. It may be better to use
`cats or monkeys as recipients, because their corneal endothe-
`lial cells more closely mimic HCECs in having little or no
`mitotic activity and a limited regenerative capacity.
`The transplanted cHCEC corneas using AM as a carrier are
`clearer and thinner than either corneas transplanted with AM
`only or corneas with removed Descemet’s membranes and
`endothelial cells. Furthermore, the transplanted cHCEC cor-
`neas are as thin as corneas with trephination only. These
`results indicate that HCECs on AM function as well as that of
`normal endothelium at least until 7 days after transplantation.
`The transplanted cHCEC corneas with AM regained partial
`transparency after transplantation. Normal corneal stromal
`clarity depends on the regular arrangement of collagen fibers,
`AM does not have such a characteristic structure. However, we
`think thinning of AM after transplantation increased its trans-
`parency, as seen in the eyes with cultured corneal epithelial
`cells transplanted for corneal epithelial diseases.10 There was a
`gap of 0.1 to 0.2 mm between donor human endothelial cells
`and host rabbit endothelial cells, but the transplanted corneas
`
`

`

`806
`
`Ishino et al.
`
`IOVS, March 2004, Vol. 45, No. 3
`
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`Erratum in: In “Quantitative Analysis of Retinal Ganglion Cell (RGC) Loss in

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