Human corneal endothelial cell transplantation with nanocomposite gel sheet preserves corneal stability in post-corneal transplant bullous keratopathy: a 16-year follow-up.

Prospects for endothelial transplantation

Human corneal endothelial cells (HCECs) are responsible for the major water -pumping function of the cornea, from corneal stroma to the aqueous humor, and any damage to these cells may lead to corneal decompensation. When the endothelium functions adequately, it regulates stromal hydration and thus both corneal thickness and transparency which are critical for optimal function. This function is accomplished by an ATP-dependent endothelial pump function, in addition to the presence of focal tight junctions which act as a barrier. HCEC density decreases with aging and with various diseases such as bullous keratopathy and Fuchs’ endothelial dystrophy. The conventional treatment for this condition is corneal transplantation using a full-thickness donor cornea. More recently an alternative surgical approach has become popular; Descemet’s stripping endothelial keratoplasty (DSEK) which selectively replaces the corneal endothelium without the need for large circumferential corneal incisions or use of sutures. Regardless of the procedure, an entire fresh and high-quality donor cornea with viable endothelium is required. However, many patients cannot receive such transplants in numerous countries due to shortage of donor corneas. Therefore, alternative sources of tissue procurement by exploiting engineering approaches to expand HCECs in vitro and fabricate transplantable sheets of HCEC would be of immense benefit. HCECs are very difficult to culture in vitro and even in the case of a successful culture, their slow replication can easily be hindered by rapidly growing stromal corneal fibroblasts (SCFs) that may have been co-isolated in some cases. Enzymatic dissociation for removing HCECs may also release stromal keratocytes into the culture system which can also turn into rapidly growing SCFs. This tissue contamination is detrimental to HCECs cultures which generally require 14 to 21 days to establish and will also adversely impact the development of tissue-engineered constructs where pure populations of cultivated and functional corneal endothelial cells are required. Furthermore, fibroblastic contamination in these cultures will interfere with the critical barrier and pump function of cultivated corneal endothelial cells. Peh et al have recently described the use of a magnetic cell separation technique to deplete contaminating SCFs from corneal endothelial cell cultures using antifibroblast magnetic microbeads.1In their study, the experimentally mixed cultures of CSFs and HCECs were tagged with antifibroblast magnetic microbeads, subjected to separation within a magnetic field, and then separated into “labeled” and “flow-through” fractions. The magnetic cell separation (MAC)-separated cells were left to adhere for at least one day to enable them to establish their morphology. Postseparation assessment of cultured fluorescent cells from both the “labeled” and “flow-through” fractions of the separated mixtures was performed to determine the efficacy of the separation. They observed a separation efficacy of 96.88% and concluded that this technique would be useful for eliminating contaminating SCFs within a culture of corneal endothelial cells.

Transplantation of Human Corneal Endothelial Cells Using Functional Biomaterials: Poly(N-isopropylacrylamide) and Gelatin

Human corneal endothelial cells (HCEC) play a pivotal role in maintaining corneal transparency. Unlike in other species, HCEC are notorious for their limited proliferative capacity in vivo after diseases, injury, aging, and surgery. Persistent HCEC dysfunction leads to sight-threatening bullous keratopathy with either an insufficient cell density or retrocorneal membrane due to endothelial-mesenchymal transition (EMT). Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy or retrocorneal membrane relies upon transplantation of a cadaver human donor cornea containing a healthy corneal endothelium. Due to a severe global shortage of donor corneas, in conjunction with an increasing trend toward endothelial keratoplasty, it is opportune to develop a tissue engineering strategy to produce HCEC grafts. Prior attempts of producing these grafts by unlocking the contact inhibition-mediated mitotic block using trypsin-EDTA and culturing of single HCEC in a bFGF-containing medium run the risk of losing the normal phenotype to EMT by activating canonical Wnt signaling and TGF-β signaling. Herein, we summarize our novel approach in engineering HCEC grafts based on selective activation of p120-Kaiso signaling that is coordinated with activation of Rho-ROCK-canonical BMP signaling to reprogram HCEC into neural crest progenitors. Successful commercialization of this engineering technology will not only fulfill the global unmet need but also encourage the scientific community to re-think how cell-cell junctions can be safely perturbed to uncover novel therapeutic potentials in other model systems.

Purpose: We previously performed human corneal endothelial precursor cell transplantation into the anterior chamber and maintenance of the prone position for 24 hours in a bullous keratopathy model. This time, we investigated the necessary postoperative time in the prone position for clinical application of precursor cell transplantation. Methods: The sphere-forming assay was used to obtain precursors from cultured human corneal endothelial cells. Chloromethyl benzamidodialkylcarbocyanine (CM-DiI)-labeled precursor cells were injected into the anterior chamber of the eye in rabbits with corneal endothelial defects, and the prone position was maintained for 0, 1, 6, or 24 hours to allow attachment to Descemet's membrane. Rabbits maintained in the prone position for 24 hours without precursor cell transplantation were the controls. Each group was observed for 28 days after surgery, followed by histological examination and fluorescence microscopy. Results: The mean corneal thickness of the rabbits kept in the prone position for 1, 6, or 24 hours after precursor cell transplantation was significantly less than that of the rabbits without adoption of the prone position after transplantation or the untransplanted rabbits at 14 days (p < 0.005), 21 days (p < 0.0001), and 28 days (p < 0.0001) after surgery. And there was no significant differences in corneal thickness between the two groups kept in the prone position for 6 hours and 24 hours throughout the observation DiI-positive human corneal endothelial-like hexagonal cells were detected on Descemet's membrane in the rabbits kept in the prone position for 1, 6, or 24 hours, but not in the control groups. Three of the six corneas in the 1-hour group showed focal edema and incomplete coverage of the endothelial defects. Conclusions: Our findings demonstrated that transplantation of human corneal endothelial precursors into the anterior chamber with adoption of the prone position for 6 hours treated bullous keratopathy in rabbits with similar efficacy to maintenance of the prone position for 24 hours after surgery.

To investigate the feasibility of transplanting untransformed human corneal endothelial cells as a treatment strategy and possible alternative for penetrating keratoplasty by growing donor cells in culture and then transplanting them to denuded Descemet's membrane of recipient corneas. Corneas from adult donors (50-80 years old) were obtained from eye banks. To grow corneal endothelial cells, Descemet's membrane with associated cells was dissected from the stroma. Endothelial cells were released by ethylenediaminetetraacetic acid treatment, grown in medium containing multiple growth factors, and identified as being of endothelial origin by morphology and by reverse-transcription polymerase chain reaction for keratin 12 and collagen type VIII. In transplantation experiments, cultured cells were seeded onto denuded Descemet's membrane of a second donor cornea at 5 x 10(5) cells/mL. The recipient cornea was incubated in organ culture for as long as 2 weeks. The morphology and ultrastructure of the endothelium were evaluated 7 and 14 days after transplantation by transmission electron microscopy, and by immunolocalization of zonula occludins-1 (ZO-1). Endothelial cell density was calculated in transplants by counting ZO-1-stained cells. Corneal endothelial cells cultured from adult donors consistently grew well in culture medium. Cells were identified as corneal endothelium by characteristic morphology and messenger RNA expression. Morphologic and ultrastructural studies of corneas containing transplanted endothelial cells demonstrated that with time there was an increase in endothelial cell-Descemet's membrane adhesion, in the extent of cell-cell contacts and lateral interdigitation, and in formation of a single cell layer. ZO-1 staining revealed tight junction formation similar to that of corneas in vivo. Mean endothelial cell density in transplanted corneas was 1,895 cells/mm(2) (range, 1,503-2,159 cells/mm(2) ). Untransformed adult human corneal endothelial cells can be efficiently and consistently cultured and transplanted onto denuded Descemet's membrane. Transplanted cells in organ culture exhibit morphologic characteristics and cell densities similar to corneal endothelial cells in vivo. These results provide evidence for the feasibility of developing methods for in vivo transplantation of untransformed corneal endothelial cells cultured from adult donor tissue.

To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells. The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.

ABSTRACTPurpose: To evaluate the effect of transplanting bioengineered corneal endothelial grafts in a rabbit model of corneal endothelial failure.Methods: Human corneal endothelial cells (HCECs) were seeded on a vitrigel carrier. After Descemet’s membrane was removed from the eyes of rabbits, transplantation was done with a vitrigel/HCEC graft or vitrigel alone without cells, or the eyes were left untreated. Slit lamp examinations and measurement of the central corneal thickness (CCT) were performed for 14 days postoperatively.Results: HCECs cultured on vitrigel were strongly positive for ZO-1 and Na+/K+ ATPase. On day 14, the cornea showed mild edema and the pupil margins were visible through the grafts in the vitrigel/HCEC graft group. HCECs completely covered the grafts on day 14. In contrast, there was severe corneal edema and the pupil margins were undetectable on day 14 after transplantation of the vitrigel carrier alone or no transplantation. Proliferation of host cells was not observed in these groups. On day 14, the mean CCT was significantly thinner in the vitrigel/HCEC graft group than in the other two groups (p = 0.0008).Conclusions: Transplantation of a vitrigel/HCEC graft was effective for reducing the corneal thickness and restoring corneal transparency, suggesting the usefulness of vitrigel as a carrier for corneal endothelial cells.

To avoid donor tissue shortages, ex vivo cultured human corneal endothelial cell (HCEC) transplantation is a promising therapeutic resource. Superparamagnetic iron oxide nanoparticle (SPION) cell labeling assists HCEC transplantation by attaching the posterior corneal stroma in ex vivo animal models. However, possible functional changes of the HCECs following SPION labeling remain to be determined. In this study, we used SPIONs to label cultured rabbit CECs (RCECs) in order to observe important cell functions and the levels of cell markers. The synthetic SPIONs exhibited superparamagnetism at room temperature, with saturation magnetization of 55.4 emu/g and negligible remanence or coercivity. The ζ-potential was -24.5 mV and the diameter was 101 ± 55 nm. Immunostaining demonstrated a normal density of zonula occluden-1 (ZO-1), nestin and Ki-67 at cellular junctions or in nuclei from RCECs following SPION labeling at 16 µg/ml. MTT cytotoxicity assay, homotypic adhesion assay, quantitative flow cytometric Ki-67 analysis and RCEC pump function measurement demonstrated no significant differences between the cells with or without SPION labeling (P<0.05, for all assays). Results of this study demonstrated successful labeled cultured RCECs with synthetic SPIONs. Labeled cells possessed several important characteristics required to maintain the transparency and refractive parameters of the cornea, including hexagonal cell morphology, higher cell adhesion ability and proliferative potential, cell pump function and the positive expression of several cell markers.

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-β signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell-cell junctions and instead activates Ras homologue gene family A (RhoA)-Rho-associated protein kinase (ROCK)-canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest-like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.

PurposeTo test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), bioengineered collagen sheets and denuded Descemet membrane (dDM) as tissue‐engineered grafts for Descemet membrane endothelial keratoplasty (DMEK).MethodsPrimary hCEC were seeded onto HALC, LinkCell™ bioengineered matrices of 20µm thickness (LK20), and dDM. Cell morphology, viability, and proliferation were assessed by light microscopy, cell viability assay, and immunohistochemistry. All hCEC‐carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea, without DM, with a regular DMEK graft as control.ResultsPrimary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96±4%) and expression of ZO‐1 and Na+/K+‐ATPase. During the in vitro surgery, hCEC‐HALC constructs behaved most similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. On the other hand, hCEC‐LK20 and hCEC‐dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC‐dDM constructs showed similar graft adherence as hCEC‐HALC constructs, while adherence of hCEC‐LK20 constructs was less effective.ConclusionsAll analyzed carriers were suitable substrates for cultivating hCEC. During in vitro DMEK surgery, hCEC‐HALC constructs behaved most similar to a standard DMEK‐graft, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC‐LK20 and hCEC‐dDM constructs.

Long-term Corneal Rejuvenation after Transplantation of Cultured Human Corneal Endothelial Cells.

Excessive loss of endothelial cells causes loss of the barrier function of the corneal endothelium, resulting in bullous keratopathy, permanent corneal clouding, and loss of visual acuity. In vivo repair of the endothelium following cell loss occurs by cell enlargement and migration, rather than by cell division. Human corneal endothelial cells (HCEC) do not divide in vivo, because they are inhibited in G1-phase of the cell cycle; however, they retain proliferative capacity. The cell cycle is divided into multiple phases. After mitogenic stimulation, cells enter G1-phase to prepare cells for DNA duplication, which occurs in S-phase. Cells then move into G2-phase to prepare cells for division, which occurs in M-phase. Movement of cells from G1- to S-phase can be prevented by the activity of the cyclin-dependent kinase inhibitors, p27Kip1, p21Cip1, and p16INK4a. These inhibitors prevent activation of the transcription factor, E2F, which is required for S-phase entry. Several factors contribute to inhibition of the proliferation of HCEC in vivo, including formation of strong cell-cell contacts (contact inhibition), lack of autocrine or paracrine growth factor stimulation, and the suppressive effect of transforming growth factor-beta2. This inhibition appears to be mediated, in large part, by p27Kip1. Although HCEC retain the ability to divide, their capacity to proliferate decreases with increasing age. This decrease is characterized by an age-related reduction in the rate of cell cycle entry and in the relative number of dividing cells. Evidence strongly suggests that this age-related decrease is the result of an up-regulation of the expression and activity of p21Cip1 and p16INK4a, but not of p27Kip1. HCEC can be induced to divide by overcoming or bypassing G1-phase inhibition using molecular biological approaches. The most promising approach so far is ectopic expression of the transcription factor, E2F2, which increases endothelial cell proliferation in ex vivo corneas from both young ( 50 years old). Proliferative capacity and the expression of senescence characteristics are also affected by endothelial topography. Cells within the central 6.0 mm diameter of the endothelium in corneas from older donors exhibit the lowest proliferative capacity and contain the highest percentage of senescent cells. The age- and topographically related decrease in proliferative capacity observed in HCEC is not due to the presence of critically short telomeres, but appears to result from sub-lethal oxidative nuclear DNA damage. Research has led to a new hypothesis regarding the molecular basis for the age- and topographically related decrease in proliferative capacity. This hypothesis states that, with increasing age, oxidative stress increases in HCEC due to their high metabolic activity and due to chronic light exposure. This results in a gradual increase in oxidative nuclear DNA damage, which leads to a decreased ability to divide, mediated by the G1-phase inhibitors, p21Cip1, and p16INK4a. This new hypothesis provides the basis for further exploration of the molecular mechanisms under-lying the age- and topographically related decrease in proliferative capacity of HCEC. This exploration could lead to the development of methods to prevent or reverse the effects of oxidative stress on these cells, thereby increasing their ability to divide in order to repair the endothelial monolayer and prevent the devastating effect on vision of the loss of endothelial barrier function.

Human corneal endothelial cells (HCEnCs) are responsible for maintaining the transparency of the cornea. Damaged or diseased HCEnCs may cause blindness. Replacement of the diseased cells with a healthy donor endothelium is the only currently available treatment. Tissue-engineering can serve as an alternative to conventional donor corneal transplantation. Due to the global shortage of donor corneas, a wide interest in the development of cultured graft substitutes and artificial corneas has increased. Availability of the old donor corneas is higher especially for research. Although it can be proposed as a valuable source for cell culture, its less proliferative capability emerges a challenge for the researchers. This article describes the use of hyaluronic acid (HA) in combination with Rho-kinase inhibitor (ROCK) Y-27632 for the cultivation of HCEnCs from older donor corneas (age > 60 years). Four conditions including and excluding HA + ROCK and its effect on early attachment rates and proliferation was studied on forty-eight corneas. It was observed that HCEnCs reach confluence within 10–15 days when cultured with HA + ROCK. This approach improves the efficiency of cell adhesion due to force attachment. HCEnCs from old donor corneas can be cultured using this method which may further lead to cell-based therapy for treating corneal endothelial dysfunction.

Corneal endothelial regeneration and tissue engineering