Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n?=?12) and fibroblastic-like (thin and very elongated cells, n?=?6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in?vitro studies of this disease.     

Postnatal development of corneal endothelial cells in normal children

Fifty-five eyes of normal children from 2 days to 11 years old were examined with specular microscopy image analyzing system, and the mean cell density (cells/mm2), mean cell area (micron2), coefficient of variation (S.D./mean), percentage of hexagonal cells, and total endothelial cell counts were calculated. The mean cell density, which statistically decreased with age varied from 5305 to 3067. The mean cell area varied from 189 microns2 to 331 microns2. Coefficient of variation and percentage of hexagonal cells varied from 0.13 to 0.26, and from 91 to 72, respectively. Generally children showed lower endothelial cellular pleomorphism and irregularity. Estimated total endothelial cell counts, in consideration of corneal enlargement, varied from 297,000 to 554,000, and showed no remarkable decrease with age. This suggested that the main reason for postnatal decrease of cell density was not cell loss but corneal enlargement.     

Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats

PURPOSE: The feasibility of corneal reconstruction with cultured adult human corneal endothelial cells (HCEC) was examined in a nude rat model. METHODS: Endothelial cells were removed from the corneas of Lewis rats using a sterile cotton swab. Cultured adult HCEC labelled with a fluorescent marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) were seeded onto the denuded Descemet's membrane. Then the corneas were centrifuged, incubated for 2 days, and transplanted into the eyes of nude rats using the penetrating keratoplasty technique (HCEC group). Control nude received corneas denuded of endothelium and without HCEC. The operated eyes were observed for 28 days after transplantation, and then were subjected to histological and fluorescein microscopic examination. RESULTS: The mean corneal thickness was significantly smaller in the HCEC group than in the control group throughout the observation period. The corneal endothelial cell density of the grafts at 28 days postoperatively ranged from 2425 to 3250 cells mm(-2) (mean+/-sd, 2744+/-337 cells mm(-2)). Fluorescein microscopy at 28 days after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the HCEC group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet's membrane. CONCLUSIONS: Cultured adult HCEC function well and maintain corneal transparency for 1 month after transplantation in nude rats.     

Morphometric analysis of retinal vascular endothelial cells in rats

INTRODUCTION: In the present study, we observed the transformation of endothelial cells in the retinal vessel of normal rats and examined the relationship between morphometrical parameters and vessel calibers. MATERIALS AND METHODS: Retinal vessels of male Wistar-Kyoto rats were stained with an en face silver staining method and the vessel caliber, the axis of the cell oriented parallel to the longitudinal axis and to the transverse plane of the vessel, and the deviation angle of endothelial cells against the vessel axis were measured under a light microscope. RESULTS: As the vessel caliber increased, endothelial cells showed a tendency to extend along the longitudinal axis in the arterioles, but endothelial cells remained unchanged in the venules. The deviation angle of endothelial cells was nearly parallel to the vessel axis especially in the arterioles. CONCLUSIONS: Change in shape of endothelial cells in the retinal arterioles suggested a high shear stress in the large arterioles, and lower shear stress in the smaller arterioles with its decrease of caliber. In the venules, however, the cell shape was unchanged, and this suggests that the blood flow is fairly steady.     

Proliferation of corneal epithelial and endothelial cells in the trabecular region of human donor corneas in organ culture

Thirty corneoscleral rings obtained after trephination of a 4- to 7.5-mm graft and 8 whole corneas with scleral rims were cultured up to 6 weeks. The corneal epithelium grew over the scleral rim and trabecula, and, after 3-4 weeks, was slowly invading the endothelial sheet. Endothelial cells did not show any evidence of migration unless the trabecula was removed. Even then, however, the endothelial growth was very limited and was soon suppressed by fast-growing epithelium.     

A histomorphometric study of corneal endothelial cells in normal human fetuses

The purpose of this study was to investigate the histomorphometric change in the normal development of human fetal corneal endothelial cells. Eighty one human fetal corneas, ranging from 12 to 40 weeks of gestation, were examined. For determination of gross parameters, corneal diameter and height were measured. Then the corneal endothelium including Descemet's membrane was stained with hematoxylin-eosin using a flat preparation method. In addition to histologic examination under the light microscope, computer-assisted image analysis was performed to determine the cell area, coefficient of variation in cell area and cell density, in both central and peripheral cornea, from each specimen. Total cell count per cornea was obtained by multiplying endothelial cell density by corneal surface area. Linear and nonlinear regression analysis of gestational age and each parameter were used to model corneal endothelial development during the prenatal period. Fetal cornea grows rapidly throughout the prenatal period. During the same period, mean cell area and total cell count also increases gradually, but there is a steep increase in the total cell count in the early period and of the cell area in the late period. The mean cell density decreases rapidly from 16 015 to 6167 cell x mm(-2). There was no significant difference in all parameters except cell density, between the central and peripheral cornea and the difference in cell density was only 2%. In the early prenatal period, there is a rapid increase of total cell count by mitosis, whereas in the late period enlarged endothelial cells cover the rapidly widening inner corneal surface without a significant change in the total cell count.     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

Efficiency of cytokine gene transfer in corneal endothelial cells and organ-cultured corneas mediated by liposomal vehicles and recombinant adenovirus

PURPOSE: Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro. METHOD: To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRalpha-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA. RESULTS: For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 microg/ml vs. 95 pg/ml per quarter of cornea). CONCLUSION: For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.     

Effects of EGF and indomethacin on rabbit corneal endothelial wound closure in excised corneas

Corneal endothelial cells of rabbit corneas stored in M-K medium at 37 degrees C were wounded by touching them lightly with a micropipet under video specular microscope observation. Three groups were studied: control, with EGF, and with EGF + indomethacin. The wound closure process (initial wound area about 8500 microns 2) was observed and recorded with time-lapse videography for 6 hr. The recorded video images were digitized and computer assisted morphometric analysis was performed. (1) Addition of either EGF (10 ng/ml) + indomethacin (1 microM), or EGF (10 ng/ml) alone to the M-K medium statistically significantly shortened the wound closure time as compared with the control group. (2) Both EGF + indomethacin and EGF alone resulted in a greater average percent relative change of the shape factor, more than three times greater with EGF + indomethacin and more than two times greater with EGF alone, than in the control group 150 min after wounding. (3) The maximum cell shape change occurred at about 150 min after wounding in the groups EGF + indomethacin and EGF alone, and at about 200 min in the control group. After this time in all three groups the cells began to approach a normal shape. (4) The cells near the wound boundary moved faster in the EGF + indomethacin and the EGF groups as compared with the control group. These results suggest that EGF and indomethacin may be of therapeutic value in promoting closure of traumatized human corneal endothelium.     

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells

BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

微机操作者与接触镜配戴者角膜内皮细胞形态学计算机分析

探讨微机操作者和接触镜（CL）配戴者角膜内皮细胞（CEC）变化及其意义。对76例微机操作者和42例CL配戴者进行了活体CEC显微镜观察和计算机形态定量分析。CL配戴者CEC密度正常，变异系数、周长、长径明显增大，形状系数减小，微机操作者各形态定量指标无明显改变。微机操作可能不会引起眼部器质性病变，而CL配戴者CEC处于不稳定状态，在其RK及内眼手术前后密切观察CEC形态定量变化十分重要。     

Ⅰ型前弹力层角膜营养不良患者角膜与正常角膜蛋白的差异表达

目的 探讨中国人Ⅰ型前弹力层角膜营养不良(corneal dystrophy of Bowman laver type 1,CDB1)患者角膜与正常角膜的蛋白差异表达.方法 取1个CDB1家系3例患者外周血,提取基因组DNA,PCR扩增TGFBI基因第4、11、12、14外显子片段并进行直接测序,证实其突变位点.取板层或穿透性角膜移植术后的病变角膜,部分标本行HE染色、阿辛蓝、PAS、刚果红、Masson三色染色后光学显微镜观察,剩余标本提取组织蛋白后进行双向凝胶电泳.以3例正常角膜组织为对照.结果 3例CDBI患者均发现TGFBI基因R124L突变.HE染色证实病变主要累及前弹力层,PAS、刚果红、Masson三色染色阳性,阿辛蓝染色阴性.在PI 4-7,相对分子质量7×103～30×103之间病变与正常角膜蛋白表达存在27个差异点.结论 中国人CDB1患者以R124L基因突变为主.CDB1角膜异常沉着性质为细胞外淀粉样纤维蛋白沉着.在PI 4-7,相对分子质量7×103～30×103之间的差异蛋白可能在CDB1发病过程中起着重要作用.     

Synthesis of glycosaminoglycans by cultures of normal human corneal endothelial and stromal cells

Monolayer cultures of normal human corneal endothelial and stromal cells were incubated with [35S]sulfate and [3H]glucosamine for 4 hr. The labeled glycosaminoglycans resulted from this incubation were isolated from the cell layer and the growth medium and further characterized. Both endothelial and stromal cell cultures synthesized a variety of sulfated glycosaminoglycans, with chondroitin 6-sulfate as the major product. Chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate were present in smaller amounts. Keratan sulfate was produced only in minimal amounts. Both cell types also synthesized hyaluronic acid. The hyaluronic acid production in stromal cell strains derived from donors of different ages was similar. The demonstration that the endothelial cell strain derived from a 1-day-old baby contained more hyaluronic acid than cultures from older donors suggests a possible age-related phenomenon as seen in developing tissues.     

Hyaluronate binding to intact corneas and cultured endothelial cells

Sodium hyaluronate (HA) protects the corneal endothelium during cataract surgery. Recently, HA receptors have been found on liver endothelial cells that play an important role in HA catabolism. It is unknown if similar receptors are present on the corneal endothelium. In this study we have used two different methods to follow the interaction of HA with corneal endothelial cells: (1) binding of 3H-HA to cells or intact corneas was determined in the presence or absence of unlabelled glycosaminoglycans after solubilization with KOH, and (2) the HA-binding region of bovine cartilage proteoglycan was used as a histochemical probe and visualized by an avidin-biotin method. 3H-HA bound both to intact rat corneas pretreated with Streptomyces hyaluronidase and to cultured monkey corneal endothelial cells. The fraction-bound 3H-HA increased with time and was saturable. Cultured endothelial cells were estimated to have 1700-2100 binding sites per cell with a binding constant of 5.6-8.5 X 10(9) liters/mol. Furthermore, unlabelled HA displaced the tritiated in a dose-dependent manner and the displacing efficiency was dependent on molecular weight. The histochemical method disclosed that HA forms a continuous layer on the endothelium. If Healon was injected into the anterior chamber, the thickness and staining intensity of this layer increased conspicuously.     

Expression and activity of cell cycle-regulatory proteins in normal and transformed corneal endothelial cells.

Corneal endothelial cells have a limited capacity for proliferation. Upon transformation with the SV40 large T antigen, however, these cells undergo division and grow rapidly. In order to gain insight into the control mechanisms that determine this proliferative switch, we investigated the expression level and activity of various known cell cycle-regulatory proteins in these cells. Primary human and rabbit corneal endothelial cells were transduced in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulatory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase correlated with the elevated expression of various cyclin-dependent kinase subunits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. Furthermore, the expression of two cyclin-dependent kinase inhibitors, p21(WAF1) and p27(KIP1), which was high in primary human cells (but not in primary rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal endothelial cells appears to be regulated at two levels: the expression of certain cell cycle-regulatory proteins that are essential for cell cycle progression is extremely low (cyclin A) or somewhat low (cdk2 and cdk4); but the amount of p21 and p27, inhibitors of cell cycle progression, is very high. As a consequence, the enzymatic activity of cyclin-dependent kinase is below detectable levels. However, the growth-inhibitory status of these components is clearly reversible: upon transduction with large T antigen, the expression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expression of p21 and p27 is inhibited, and the cells proliferate. Thus, our study provides insight into the molecular basis of the attenuated proliferation of corneal endothelial cells and suggests potential targets that could be manipulated for the purpose of therapeutic interventions aimed at renewed cell growth.     

Morphometric analysis of corneal endothelium following radial keratotomy

We performed morphometric analysis of the central corneal endothelium on 24 eyes of 19 patients who had had anterior radial keratotomy. The endothelium was analyzed for a variety of parameters, including cell area, perimeter, side lengths, cell shape, and number of sides. Mean, standard deviation, and coefficient of variation were calculated for each parameter. The mean cell density decreased from 2,835 to 2,677 cells/mm2, mean cell perimeter increased from 71.4 micron to 74.3 micron, and mean side length increased from 11.8 micron to 12.3 micron following surgery. The changes in these three parameters were statistically significant (P less than 0.05). The coefficient of variation of cell area (polymegathism) changed from 0.319 to 0.307, the hexagonality changed from 62.5% to 59.6%, and the cell shape changed from 0.872 to 0.867. The changes in these parameters were not statistically different before and after surgery. The group of patients that had no reported microperforations showed only a small decrease of cell density (1.6%), while the group of patients that had microperforations showed a large decrease of cell density (14.3%). The cell perimeter and side lengths showed a similar pattern. The group of corneas with the optical zone diameter less than 3.5 mm showed a decrease in mean cell density from 2,994 to 2,725 cells/mm2, and the cell shape changed from 0.874 to 0.866 following surgery. The changes in these parameters were statistically significant (P less than 0.05) before and after surgery. Among all factors associated with radial keratotomy, microperforation and a small diameter of central optical zone appear to be the two greatest risk factors.     

Transplantation of corneal endothelial cells

Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells. In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs). We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum. We performed the following analysis utilizing these cultured HCECs. The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs. The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting. HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases. Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages. These results indicated that shortening of telomere length is not related to senescence of HCECs. We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs. The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs. HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions. Primary and passage culture of HCECs was possible using adult human serum. We reconstructed the cornea using cultured HCECs and human corneal stroma. The corneal stroma, on which the cell suspension of HCECs was poured, was mildly centrifuged to enhance the HCECs attachment to the stroma. The cell density of HCECs on the reconstructed cornea reached 2,500 cells/mm2. The pump function of the reconstructed cornea was measured with an Ussing chamber. The potential difference in the reconstructed cornea and normal cornea was 0.30 mV and 0.40 mV, respectively; indicating that the pump function of the reconstructed cornea is 75% of that of the normal cornea. The reconstructed cornea was transplanted to a rabbit eye and stayed transparent for 6 months after the operation. Fluorescein labeled cultured HCECs remained on the graft 1 month after the transplantation, indicating that transplanted HCECs contributed to the transparency of the graft. The possibility of using artificial stroma or porcine corneal stroma as a carrier of cultured HCECs was investigated. The artificial stroma made of alkaline-treated collagen could not be sutured but showed good transparency, biocompatibility, and cell-attachability. Porcine corneal stroma, expressing little xeno-sugar antigen alpha-gal epitope, induced no super acute rejection but mild cellular rejection when transplanted in the cornea of animals possessing natural antibody to alpha-gal epitope. The cornea reconstructed with porcine corneal stroma and HCECs had an average cell density of 1721/mm2 and had approximately 60% of the pump function of a normal cornea. As new technologies in corneal transplantation, the application of self immature cells and the direct delivery of cultured HCECs into the anterior chamber were investigated. Part of rat mononuclear cells that were obtained from the bone marrow and injected into the rat anterior chamber transformed into corneal endothelium-like cells, suggesting that self immature cells can transform into corneal endothelial cells. Cultured rabbit corneal endothelial cells that endocytosed iron were injected into the anterior chamber of rabbits whose corneal endothelium was cryo-injured, and were pulled to Descemet's membrane by putting a magnet on the eyelid. In these rabbits, corneal edema decreased more quickly than in the control group and no intraocular pressure rise was observed during 8 weeks after the operation, suggesting that the direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice. The following obstacles should be addressed to make the transplantation of cultured corneal endothelial cells clinically applicable. 1. To reconstruct a cornea that is the same as or superior to the normal cornea, more innovation is necessary in the method of culturing and seeding HCECs. We should consider utilizing HCECs obtained from fetuses after clearing ethical issues. Moreover, we need to develop a method to enhance the cell density and the cell functions. 2. Porcine corneal stroma is promising as a carrier of HCECs instead of human corneal stroma, which is in very limited supply. The usefulness of porcine corneal stroma acellularized to prevent retrovirus infection should be evaluated. 3. To make the self immature cells applicable to corneal transplantation, we should elucidate the corneal endothelial cell specific markers and the factors that are necessary to induce self immature cells to become corneal endothelial cells. 4. The direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice when its long-term safety is confirmed.