Heterologous transplantation versus enhancement of human corneal endothelium

The ability to successfully transplant human corneal endothelium would offer a significant advance in the treatment of many corneal diseases. To investigate the feasibility of this, we established cultures of endothelial cells derived from neonatal human corneas. Eye bank donor corneas were either enhanced with a suspension of cultured endothelial cells or underwent endothelial cell removal and subsequent replacement with cultured endothelium. Following a 48-h incubation, the corneas were transplanted into the eyes of nonhuman primates. Over a 12-month period, 67% of the corneas with complete endothelial cell replacement thinned and remained clear, with a mean corneal thickness of 0.57 mm. Enhanced corneal buttons demonstrated a significantly lower success rate (35%), with opacified and thickened corneas. Control eyes in which the native endothelium was removed demonstrated advanced corneal edema and vascularization, with a mean corneal thickness in excess of 1 mm. By utilizing established tissue-culture techniques, we have demonstrated that human corneal endothelium, when cultured and subsequently transplanted, retains its in vivo pump function. Although further studies are warranted, these results indicate that transplanted human corneal endothelial cells can function normally and suggest the possibility of endothelial cell replacement for therapeutic purposes.     

重组牛碱性成纤维细胞生长因子对角膜内皮细胞密度低下患者白内障术后角膜内皮的保护作用

目的　研究角膜内皮细胞密度（ECD）低下患者行超声乳化白内障吸除术后应用重组牛碱性成纤维细胞生长因子（re-bFGF）对角膜内皮的保护作用。方法　前瞻性队列研究。选取2019年9月至2022年4月在北京大学第三医院眼科就诊、拟行超声乳化白内障吸除且合并ECD低下的患者80例 (90眼)为研究对象,随机分为两组,试验组41例(45眼),对照组39例(45眼)。术后除常规抗炎治疗外,试验组患眼应用re-bFGF滴眼液每日4次滴眼,对照组患眼应用1 g·L^-1玻璃酸钠滴眼液每日4次滴眼,均使用至术后6个月。对比分析两组患眼术前和术后1个月、3个月、6个月的ECD和中央角膜厚度（CCT）等。结果　术后1个月、3个月、6个月,试验组患眼的ECD和CCT均较术前变化不明显（均为P>0.05）,而对照组患眼的ECD均较术前下降,CCT均较术前增加（均为P<0.05）。术前,试验组和对照组患眼ECD分别为（1120.6±306.1）个·mm^-2、（1040.5±317.3）个·mm^-2 ,CCT分别为（543.1±51.6）μm、（546.8±35.6）μm,两组相比差异均无统计学意义（均为P>0.05）;术后6个月,试验组和对照组患眼ECD分别为（1271.3±288.6）个·mm^-2、（746.5±193.5）个·mm^-2 ,CCT分别为（542.0±55.3）μm、（583.5±45.3）μm,两组相比差异均有统计学意义（均为P<0.05）。术后2眼发生了角膜内皮失代偿,且均发生在对照组。结论　re-bFGF对ECD低下患者行超声乳化白内障吸除术后的角膜内皮有保护作用,可减轻超声乳化手术造成的ECD下降,减少角膜水肿及术后短期角膜内皮失代偿的严重并发症的发生率。     

Evaluation of corneal endothelium after pediatric cataract surgery in children and adolescents

PURPOSE: to evaluate the effects of cataract extraction and lens implantation on corneal endothelium morphology. MATERIAL AND METHODS: In 21 eyes of 14 children with congenital or developmental cataract, corneal endothelium was studied. Patient age was 9 to 19 years (mean 12.9 years). In all eyes extracapsular cataract extraction (ECCE) with PMMA intraocular lens implantation was performed, without primary posterior capsulotomy or anterior vitrectomy. Lens wearers, patients with traumatic cataract or external eye diseases and ocular surgery in history were excluded. The endothelium was imaged by non-contact microscope Topcon SP-2000P. This examination was done preoperatively and 1 month, 6 months and 1 year postoperatively. Corneal thickness (T), corneal endothelial density (ECD) and mean cell area (AVG) of endothelial cells were examined. RESULTS: Mean corneal thickness was 0.55 mm after 1 month, 0.54 mm after 6 months and 0.54 mm after 12 months. The mean preoperative endothelial cell density was 3231.1 cells/mm2. ECD after 1.6 and 12 months was 2874.3; 2639.2 and 2479.9 cells/mm2 respectively. Mean endothelial cell loss was 10.94% after 1 month, 17.85% after 6 months and 22.68% after 12 months. AVG before operation was 315.8 mm2, after 1 month 355.8 mm2, after 6 months 382.4 mm2 and 399.5 mm2 after 12 months. CONCLUSIONS: Changes in corneal endothelium morphology had no effect on transparency of the cornea.     

硅油对角膜内皮的影响

目的：探讨硅油在不同情况下对角膜内皮的影响。方法;对１７０只眼行不同手术术前,术后２周,４周,８周及３个月时的角膜内皮细胞密度进行观察记录,同时对硅油人前房的２０只眼进行观察。结果;在眼压控制良好的情况下,有晶体眼行单纯玻璃体和切割术及玻璃体切割硅的填充术,无晶体眼行玻璃体切割硅油填充术。有晶体眼及无晶体眼行硅油取出术手术后各期内皮细胞密度与术前相比均无显著差异,有晶体眼行玻璃体切割晶体切割硅油填     

Outcomes of penetrating keratoplasty with imported donor corneas

PURPOSE: To analyze factors influencing the surgical success of penetrating keratoplasty and long-term graft survival when using imported donor corneas. METHODS: Sixty-three donor corneas imported to Taipei from the Cincinnati Eye Bank from July 1992-June 1993 were used for penetrating keratoplasty. The corneal endothelium was examined using specular microscopy on arrival in Taiwan. The endothelial morphology and endothelial cell density (ECD) were compared with the photograph of the same cornea taken in the United States. The relationships of the surgical success rate with donor age, death to enucleation time, death to surgery time, and ECD were analyzed. The long-term graft survival and ECD of clear grafts were analyzed 4 years after surgery. RESULTS: On specular microscopic examination. the imported corneas showed diminished endothelial reflection, blurred cellular borders, and increased dark areas, which were markedly different from the pictures of the corneal endothelium taken in the United States. The average ECD before transportation was 2,525+/-267/mm2 and decreased to 1,934+/-250/mm2 after transportation (p < 0.001), with an average endothelial cell loss of 590+/-247/mm2. The overall surgical success rate was 89% and did not correlate with any of the donor factors tested except death to surgery time. The surgical success rate decreased when the time from death to surgery was >7 days (p = 0.05), mainly because of poor reepithelialization. Four years after surgery, 24 grafts remained clear. The ECD had decreased by 72+/-5% in the clear grafts. CONCLUSIONS: Our findings show that endothelial changes in imported donor corneas do occur after transportation, but the surgical success rate may not be influenced significantly if the penetrating keratoplasty is performed within 7 days after donor death. However, the ECD in the clear grafts 4 years after surgery is low.     

Corneal endothelial cells 6–7 years following cataract surgery in patients with pseudoexfoliation syndrome

Purpose: To assess the condition of the corneal endothelium an extended period after cataract surgery in eyes with and without pseudoexfoliation syndrome (PES). Methods: Forty‐six patients with PES who underwent cataract surgery in the Eye Department, Oslo University Hospital, in 2001 and 2002 were enrolled and compared to 101 matched controls without PES who had surgery in the same period. They were re‐examined 6–7 years following surgery with measurements taken of corneal endothelial cell density (ECD), pleomorphism, polymegathism and corneal thickness. Results: Mean ECD was 2024 ± 371 cells/mm² in eyes with PES and 2144 ± 365 cells/mm² in eyes without PES. The difference was not statistically significant. No significant difference in polymegathism and pleomorphism was noted. Mean corneal thickness was 543 and 547 μm in eyes with and without PES, respectively (not statistically significant). The presence of glaucoma in pseudoexfoliative eyes was not associated with endothelial cell changes. Conclusion: Six to 7 years following cataract surgery, no statistically significant differences were established in ECD, pleomorphism, polymegathism and corneal thickness in eyes with and without PES. No clinical signs of corneal decompensation were noted amongst the participants.     

Extended incubation times improve corneal endothelial cell transplantation success

To investigate the ability of extended incubation times to improve the success of endothelial cell transplantation, eight human donor corneas were denuded of their native endothelium, seeded twice during a 1-hr interval with a suspension of cultured infant human corneal endothelial cells, and then incubated for 144 hr under standard conditions. Subsequently the corneas were transplanted into African green monkeys using routine penetrating keratoplasty techniques. Rotational autografts and corneas devoid of endothelial cells served as controls. The seeded corneas appeared hazy at the time of surgery (mean pachymetry 48 hr postoperatively, 0.794 mm). Six corneas (75%) subsequently cleared, yielding a mean corneal thickness of 0.541 +/- 0.040 and 0.554 +/- 0.040 at 6 and 12 postoperative months, respectively. All control eyes showed advanced edema (thickness, greater than 1.0 mm) and developed extensive neovascularization. Clinically, the extended postseeding incubation corneas were observed to clear more rapidly and stabilize their thickness earlier than corneas incubated for only 24-48 hr. Scanning electron microscopy of extended postseeding incubation corneas revealed an intact monolayer of contact-inhibited cells with the hexagonal mosaic typical of corneal endothelium in vivo and improved intercellular contact compared with corneas incubated for only 24-48 hr.     

角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

利用后弹力层撕除术建立兔眼角膜内皮失代偿模型的评估

目的利用后弹力层撕除术建立一种新的角膜内皮失代偿模型以便更好地了解该手术的组织反应。方法根据手术方法的不同将40只新西兰成年兔平均分为4组：角膜内皮刮除组、后弹力层撕除组、后弹力层撕除角膜内皮移植术（DSEK）组及DSEK供体组;右眼为手术眼。每组定期通过角膜内皮活体染色,眼前节照相和UBM至少观察2个月。结果后弹力层撕除组角膜始终保持混浊,角膜内皮刮除组和DSEK组角膜逐渐透明,角膜厚度逐渐降低。活体染色显示角膜后弹力层撕除组术后2个月仍无角膜内皮生长。结论后弹力层撕除术建立的角膜内皮失代偿模型显示了后弹力层撕除后角膜内皮愈合过程,可用于角膜内皮移植的研究。     

Corneal endothelium and postoperative outcomes 15 years after penetrating keratoplasty

PURPOSE: To determine changes in the central endothelium and thickness of grafted corneas and the cumulative probability of developing glaucoma, of graft rejection, and of graft failure 15 years after penetrating keratoplasty. DESIGN: Longitudinal cohort study of 500 consecutive penetrating keratoplasties by one surgeon. METHODS: Regrafted eyes, fellow eyes of bilateral cases, and patients not granting research authorization were excluded, leaving 388 grafts for analysis. At intervals after surgery, we photographed the endothelium and measured corneal thickness using specular microscopy. The presence of glaucoma, graft rejection, and graft failure were recorded. RESULTS: The 67 patients examined at 15 years represented 30% of the available clear grafts. Endothelial cell loss from preoperative donor levels was 71 +/- 12% (mean +/- standard deviation, n = 67), endothelial cell density was 872 +/- 348 cells/mm(2), and corneal thickness was 0.59 +/- 0.06 mm. Endothelial cell density was unchanged between 10 and 15 years, whereas corneal thickness increased (P = .001, n = 55). The mean annual rate of endothelial cell loss from 10 to 15 years after surgery was 0.2 +/- 5.7% (n = 54). The cumulative probability of developing glaucoma, graft rejection, or graft failure was 20%, 23%, and 28%, respectively, and 6 of the 8 graft failures after 10 years resulted from late endothelial failure. CONCLUSIONS: From 10 to 15 years after penetrating keratoplasty, the annual rate of endothelial cell loss was similar to that of normal corneas, corneal thickness increased, and late endothelial failure was the major cause of graft failure.     

Evaluation of grafted patients with donor corneas that today are more than 100 years old

Purpose: Life expectancy is increasing. When corneal donors become older and corneal‐grafted patients live longer with their graft, the need for good‐quality donor tissue becomes more crucial. The aim of the present investigation is to study grafted recipients with a donor cornea with a total tissue age of more than 100 years. Methods: One thousand consecutive donor records from the Danish Cornea Bank were initially reviewed. After applying different inclusion criteria, 35 recipients with corneal donor tissue of more than 100 years of age were invited for a follow‐up visit. Visual acuity, corneal transparency and thickness, and intraocular pressure were measured. Corneal topography and endothelial photos were taken. Results: Seventeen of the invited patients attended the examination. The average age of the grafts at examination was 107 years old; the oldest being 118 years. Most grafts were still clear 23–35 years after transplantation, and almost one‐fourth had best spectacle‐corrected visual acuity (BSCVA) ≥ 0.50. Cell morphology showed irregularity in size and shape for both grafted and healthy corneas, but the alterations were more extensive in the grafted corneas. The average endothelial cell density (ECD) was 1360 mm² in the grafted corneas. Sixty per cent had ECD > 1000 cells/mm². No signs of decompensation were observed for those with <1000 cells. The average central corneal thickness of the grafts was 0.582 mm (SD = 0.067) compared with 0.494 mm (SD = 0.043) in the fellow cornea. Conclusion: This study shows a trend of moderate long‐term survival and quality for very old grafts despite low ECD. Most recipients had a clear transplant, and one‐fifth had BSCVA of 0.80.     

Effects of posterior chamber lens implantation on the endothelium of transplanted corneas

AIMS—The morphological changes of the corneal endothelium after posterior chamber lens implantation in the transplanted corneas were investigated.
METHODS—36 patients underwent extracapsular cataract extraction with posterior chamber lens implantation. Among these, penetrating keratoplasty had been performed in 18 patients before cataract surgery. The indications for penetrating keratoplasty in these cases included keratoconus, herpetic keratitis, and macula cornea. 18 cataract patients with normal corneas were also studied as controls. The central corneal endothelium in each subject was examined with a wide field specular microscope at a few days before and 3 months after cataract surgery.
RESULTS—Although the transplanted corneas showed lower endothelial cell densities, marked polymegethism, and pleomorphism in the baseline variables, the endothelial morphological changes in the transplanted corneas after posterior chamber lens implantation were comparable with those in the normal corneas. Also, there was no clinical evidence, especially, of corneal epithelial and/or endothelial rejections and corneal decompensation in all corneas.
CONCLUSION—Even though the transplanted corneas have a lower endothelial cell density and marked polymegethism, it is believed that cataract surgery does not induce corneal decompensation in cases where the peripheral recipient endothelium can be considered to have normal morphology.

     

Transplantation of cultured human neonatal corneal endothelium

Human neonatal corneal endothelial cells were successfully maintained in tissue culture, morphologically resembling adult corneal endothelium. Eyebank donor corneas were obtained, denuded of their native endothelium and seeded with a suspension of the cultivated neonatal endothelial cells. After 48 hours, the eye-bank tissue was then transplanted into the eyes of Rhesus monkeys. Over a five month period, five of eight transplants cleared, with a mean central corneal thickness of 0.480 mm and endothelial cell densities ranging from 560 to 1650 cells/mm2. All control eyes without donor endothelium remained cloudy. In the experimental group three eyes initially thinned but subsequently became edematous. Further studies are needed to improve the seeding procedure and to assess the long-term viability of transplanted endothelium.     

Increased endothelial cell density in the paracentral and peripheral regions of the human cornea

PURPOSE: To systematically investigate the central, paracentral, and peripheral endothelial cell density (ECD) in normal human corneas. DESIGN: Observational case series and experimental study. METHODS: Noncontact specular microscopy was undertaken to determine the ECD of the central, paracentral (2.7 +/- 0.2 mm from center) and peripheral (4.7 +/- 0.2 mm from center) regions of the cornea of 48 normal eyes. The ECDs of central and peripheral regions were also determined with contact specular microscopy in 21 normal eyes and a group of 30 Optisol-GS eye bank corneas were evaluated with alizarin red stain. Histologic ECD of 13 Optisol-GS stored corneas were also determined. RESULTS: Paracentral and peripheral ECD measured with the noncontact specular microscope were 5.8% (P <.01) and 9.6% (P <.001) increased compared with central ECD. Superior peripheral ECD was increased compared with the other three peripheral quadrants (P <.05) and was 15.9% higher than central ECD. Contact specular microscopy showed an increase of 8.9% in the peripheral ECD from the center. Alizarin red stained corneas confirmed the specular microscopy numbers with a 9.2% increase in the paracentral region, and a 17.2% increase in the peripheral region. Histological cross sections of human corneas also showed a 22.9% increase in peripheral ECD compared with the central region. CONCLUSIONS: The human cornea has an increased ECD in the paracentral and peripheral regions of cornea compared with the central region. The superior peripheral region of the corneal endothelium has the largest increase in ECD. These data on normal endothelial cell distribution in the human cornea are especially significant as they relate to new surgical techniques and endothelial wound repair.     

Morphometric analysis of corneal endothelial giant cells in normal and traumatized corneas

Corneal endothelial cells from normal and traumatized human, primate, cat and rabbit eyes were studied by specular microscopy. Morphometric analysis was performed on micrographs of corneal endothelium using a semi-automated image analysis system. The results showed that under normal conditions the corneal endothelium of all four species exhibit major morphological similarities (mean cell areas: human 317 ± 32 μm², primate 246 ± 22 μm², cat 357 ± 25 μm², rabbit 308 ± 35 μm²). The normal corneal endothelium in man was found to be more polymegethous than that of the other species. Trauma to cat, primate and human corneas resulted in a long-term reduction in endothelial cell density and enhanced polymegethism. In contrast, the reparative response of the rabbit ensured the reformation of an essentially normal monolayer following injury. Endothelial giant cells were a normal inclusion in the rabbit corneal endothelium but were only significant in cat, primate and man following trauma. The presence of corneal endothelial giant cells in amitotic corneas may therefore represent a compensatory response in the absence of mitotic potential.     

Cornea procurement from very old donors: post organ culture cornea outcome and recipient graft outcome

AIM: To study the suitability of corneas from very old donors for graft after banking and their clinical and endothelial outcomes in recipients. METHODS: 419 corneas stored in organ culture were divided into group 1, donors under 85 years (330 corneas) and group 2, "very old" donors aged 85 years and over (89 corneas). Endothelial cell density (ECD) before and after organ culture, discard rate before and after storage, and clinical and endothelial outcomes of the 196 penetrating keratoplasties (PKP) (158 in group 1 and 38 in group 2) were compared in a prospective longitudinal study. RESULTS: Initial ECD was lower in group 2 than in group 1 and elimination for low ECD was more frequent in group 2 (respectively 38% v 20.2%, p=0.001). At the end of storage, because very old corneas lost fewer ECs than younger ones (respectively 4.2% v 9.5%, p=0.022), ECD was comparable between the two groups. The corneas of very old donors had a poorer macroscopic appearance at procurement and during surgery. Despite this, in grafted patients, overall graft survival in groups 1 and 2 (respectively 87.4% v 80.6%, p=0.197), visual acuity, and ECD did not differ at completion of the study (mean follow up 25 months). CONCLUSION: This study suggests that endothelial cell count during banking ensures that functional and cellular results of PKPs are not dramatically influenced by very old donor age. Considering Europe's ageing population, the very elderly should not be deemed off limits for corneal procurement.     

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PURPOSE: To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. METHODS: Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. RESULTS: Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. CONCLUSIONS: Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.     

Early corneal edema following topical application of mitomycin-C

PURPOSE: To determine the effect of mitomycin-C (MMC) on the cornea after a single intraoperative application. SETTING: Department of Ophthalmology, Far Eastern Memorial Hospital, Ban-Chiao, Taipei, Taiwan. METHODS: Mechanical epithelium debridement of the central 10.0 mm of the cornea was performed in 63 pigmented rabbits. One group of corneas (MMC1, n = 42) was soaked with MMC 0.01% solution for 2 minutes; the second group (MMC2, n = 42) was soaked with MMC 0.02% solution for 2 minutes. Control corneas (n = 42) were soaked with balanced salt solution for 2 minutes. Changes in the central corneal thickness, clarity, epithelial defect size, endothelial cell density, and endothelial apoptosis in the 3 groups were examined on days 0, 1, 2, 3, 5, 7, and 14. RESULTS: There was a dose-dependent increase in corneal thickness, decrease in corneal clarity, and increase in endothelial apoptosis after a single intraoperative application of MMC. The endothelium was significantly swollen and became pleomorphic and polymegethic with a concomitant decrease in endothelial cell density, also in a dose-dependent manner. CONCLUSIONS: A single application of MMC on the corneal surface caused dose-dependent corneal edema and endothelial apoptosis in the rabbit model. Further clinical study of human eyes is warranted.     

Expression of adhesion molecule CD44 on human corneas

AIMS—This study was undertaken to confirm the distribution and expression of the molecule CD44 on human corneas under normal and pathological conditions.
METHODS—Fifty eight corneal buttons from adult patients suffering from various corneal diseases and four normal corneas were included in this study. Frozen sections were stained immunohistochemically with monoclonal antibodies against human CD44 using an APAAP method and observed under a light microscope.
RESULTS—In normal corneas CD44 was predominantly expressed on the membranes of basal epithelial cells and on the keratocytes, as well as on the vascular endothelial cells of the corneal limbi, but was not expressed on corneal endothelial cells. Enhanced expression of CD44 was observed on the epithelium of corneas with inflammation and allograft rejection. In a number of abnormal conditions including allograft rejection, corneal trauma, primary and secondary corneal endothelial decompensation the remaining endothelial cells stained positively for CD44. However, in some corneas of keratitis, keratoconus, and dystrophy the endothelium which appeared relatively integral in morphology and amount remained CD44 negative.
CONCLUSIONS—These results suggest that CD44, the hyaluronate receptor, may play an important role in corneal cell-cell and cell-matrix interactions. Its regulation is closely related to corneal inflammatory reactions. The induction of CD44 on corneal endothelium might play a potential role in compensatory processes when corneal endothelial cells are injured.

     

Corneal endothelial regeneration and tissue engineering

Human corneal endothelial cells (HCECs) have a limited proliferative capacity. Descemet stripping with automated endothelial keratoplasty (DSAEK) has become the preferred method for the treatment of corneal endothelial deficiency, but it requires a donor cornea. To overcome the shortage of donor corneas, transplantation of cultured HCEC sheets has been attempted in experimental studies. This review summarizes current knowledge about the mechanisms of corneal endothelial wound healing and about tissue engineering for the corneal endothelium. We also discuss recent work on tissue engineering for DSAEK grafts using cultured HCECs and HCEC precursor cell isolation method (the sphere-forming assay). DSAEK grafts (HCEC sheets) were constructed by seeding cultured HCECs on human amniotic membrane, thin human corneal stroma, and collagen sheets. The pump function of the HCEC sheets thus obtained was approximately 75%–95% of that for human donor corneas. HCEC sheets were transplanted onto rabbit corneas after DSAEK. While the untransplanted control group displayed severe stromal edema, the transplanted group had clear corneas throughout the observation period. The sphere-forming assay using donor human corneal endothelium or cultured HCECs can achieved mass production of human corneal endothelial precursors. These findings indicate that cultured HCECs transplanted after DSAEK can perform effective corneal dehydration in vivo and suggest the feasibility of employing the transplantation of cultured HCECs to treat endothelial dysfunction. Additionally, corneal endothelial precursors may be an effective strategy for corneal endothelial regeneration.