Corneal Endothelial Cell Transplantation Research Articles

Bioprinting of human pluripotent stem cell derived corneal endothelial cells with hydrazone crosslinked hyaluronic acid bioink

BackgroundHuman corneal endothelial cells lack regenerative capacity through cell division in vivo. Consequently, in the case of trauma or dystrophy, the only available treatment modality is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor which faces a high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink.MethodsCorneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas.ResultsThe bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10 days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer.ConclusionsOur results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.

Electrospun Nanofiber Membrane for Cultured Corneal Endothelial Cell Transplantation.

The corneal endothelium, comprising densely packed corneal endothelial cells (CECs) adhering to Descemet's membrane (DM), plays a critical role in maintaining corneal transparency by regulating water and ion movement. CECs have limited regenerative capacity within the body, and globally, there is a shortage of donor corneas to replace damaged corneal endothelia. The development of a carrier for cultured CECs may address this worldwide clinical need. In this study we successfully manufactured a gelatin nanofiber membrane (gelNF membrane) using electrospinning, followed by crosslinking with glutaraldehyde (GA). The fabricated gelNF membrane exhibited approximately 80% transparency compared with glass and maintained a thickness of 20 µm. The gelNF membrane demonstrated desirable permeability and degradability for a Descemet's membrane analog. Importantly, CECs cultured on the gelNF membrane at high densities showed no cytotoxic effects, and the expression of key CEC functional biomarkers was verified. To assess the potential of this gelNF membrane as a carrier for cultured CEC transplantation, we used it to conduct Descemet's membrane endothelial keratoplasty (DMEK) on rabbit eyes. The outcomes suggest this gelNF membrane holds promise as a suitable carrier for cultured CEC transplantation, offering advantages in terms of transparency, permeability, and sufficient mechanical properties required for successful transplantation.

Трансплантация суспензии эндотелиальных клеток в эксперименте ex vivo

Relevance. Corneal transplantation with entire cornea or anormal layers replacement remains the only treatment option for patients with corneal endothelium pathology. Selective endothelial replacement was introduced into practice two decades ago, namely posterior lamellar keratoplasty, which has advantages over penetrating keratoplasty. Improvements of this method led to technique creation where isolated Descemet's membrane with endothelium monolayer without stromal layer is transplanted. This technology is associated with risks of intra- and postoperative complications, and is difficult to perform. Nowadays, development is in progress of introducing corneal endothelial cells (CECs) transplantation without stromal layer and even without Descemet's membrane. CECs transplantation in suspension form has potential to change treatment approach of corneal endothelium pathologies giving possibility of rapid eyesight recovery and reducing need for graft material. Purpose. To evaluate effectiveness of corneal CECs suspension transplantation on cadaver eye in ex vivo experiment. Material and methods. CECs suspension was obtained by modified enzymatic method. CECs transplantation experiment consisted of 3 stages. Firstly, CECs loss was calculated depending on cell administration method. Secondly, corneoscleral button was used as recipient for the obtained suspension. The third stage was carried out on cadaver eyeball. Results. The viability of transplanted CECs and their effective adhesion to Descemet's membrane was confirmed in this experiment. The characteristic markers of CECs ZO-1, Na+/K+ -ATPase, Ki67 were determined in corneal samples by immunohistochemistry assay and single cells expressing Vimentin were detected after one-week cultivation. Conclusion. Based on transplantation experiment results, it can be noted this technique is quite promising in surgical rehabilitation of patients with corneal endothelial dysfunction. Thus, it seems relevant to continue research to achieve the main goal – to fully cover the defect of central zone of corneal posterior surface with isolated CECs given that their morphology and functional activity are preserved. Key words: cornea, endothelial corneal dystrophy, endothelial cell suspension, corneal endothelial cell transplantation

Therapeutic Potency of Induced Pluripotent Stem-Cell-Derived Corneal Endothelial-like Cells for Corneal Endothelial Dysfunction.

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Characterization of Taurine/Silk Fibroin Blend Film for Application as a Carrier for Corneal Endothelial Cell Transplantation

Characterization of Taurine/Silk Fibroin Blend Film for Application as a Carrier for Corneal Endothelial Cell Transplantation

Novel Corneal Endothelial Cell Carrier Couples a Biodegradable Polymer and a Mesenchymal Stem Cell-Derived Extracellular Matrix.

Here, we report a transparent, biodegradable, and cell-adhesive carrier that is securely coupled with the extracellular matrix (ECM) for corneal endothelial cell (CEC) transplantation. To fabricate a CEC carrier, poly(lactide-co-caprolactone) (PLCL) solution was poured onto the decellularized ECM (UMDM) derived from in vitro cultured umbilical cord blood-MSCs. Once completely dried, ECM-PLCL was then peeled off from the substrate. It was 20 μm thick, transparent, rich in fibronectin and collagen type IV, and easy to handle. Surface characterizations exhibited that ECM-PLCL was very rough (54.0 ± 4.50 nm) and uniformly covered in high density by ECM and retained a positive surface charge (65.2 ± 57.8 mV), as assessed via atomic force microscopy. Human CECs (B4G12) on the ECM-PLCL showed good cell attachment, with a cell density similar to the normal cornea. They could also maintain a cell phenotype, with nicely formed cell-cell junctions as assessed via ZO-1 and N-cadherin at 14 days. This was in sharp contrast to the CEC behaviors on the FNC-coated PLCL (positive control). A function-related marker, Na+/K+-ATPase, was also identified via western blot and immunofluorescence. In addition, primary rabbit CECs showed a normal shape and they could express structural and functional proteins on the ECM-PLCL. A simulation test confirmed that CECs loaded on the ECM-PLCL were successfully engrafted into the decellularized porcine corneal tissue, with a high engraftment level and cell viability. Moreover, ECM-PLCL transplantation into the anterior chamber of the rabbit eye for 8 weeks proved the maintenance of normal cornea properties. Taken together, this study demonstrates that our ECM-PLCL can be a promising cornea endothelium graft with an excellent ECM microenvironment for CECs.

Gelatin scaffold with multifunctional curcumin-loaded lipid-PLGA hybrid microparticles for regenerating corneal endothelium

Corneal transplantation is currently the only approach to cure corneal blindness. Cell-based strategies that employ corneal endothelial cells (CECs) grown on supporting biomaterials hold great promise as possible alternative therapies for treating corneal endothelial dysfunction. Nevertheless, most biomaterials are used merely because of their robust mechanical properties, providing passive physical support for the transplantation of CEC monolayers. Based on the versatility of curcumin in ophthalmic applications, this study aims to develop a multifunctional scaffold system that can not only support the function and transplantation of CECs but also prevents post-engraftment complications by sustained curcumin release, thus enhancing the long-term success of CEC engraftment. Curcumin-loaded lipid-poly(lactic-co-glycolic acid) (PLGA; Cur@MPs) hybrid microparticles (MPs) fabricated using an oil-in-water single emulsion method are embedded into gelatin-based scaffolds. The anti-inflammatory, antioxidative, and anti-angiogenic potentials of the developed scaffolds and their capacity in supporting CEC monolayer formation are evaluated. The Cur@MPs are capable of promoting CEC proliferation, protecting CECs from oxidative stress-induced cell death via modulating Nrf2/HO-1 signaling axis, suppressing the secretion of pro-inflammatory cytokines by macrophages, and inhibiting the migration and angiogenesis of vascular endothelial cells. By incorporating the Cur@MPs into a thin gelatin membrane, the fabricated scaffold is able to support the growth and organization of CECs into a polygonal morphology with tight junctions. These experimental results demonstrate the potential of the Cur@MPs-loaded gelatin scaffold for actively supporting the survival and function of CEC monolayers after transplantation.

In vitro evaluation and transplantation of human corneal endothelial cells cultured on biocompatible carriers

AbstractPurposeTo 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.

Ultrathin, Strong, and Cell-Adhesive Agarose-Based Membranes Engineered as Substrates for Corneal Endothelial Cells.

We aimed to bioengineer a scaffold that can facilitate the transplantation of corneal endothelial cells (CEC), given the global shortage of cadaveric donor tissues. Although agarose (A) has outstanding biocompatibility and mechanical properties, it natively does not permit cell adhesion. In this study, agarose was modified with different attachment signals: GRGD (giving AR as product), lysine (AK), poly lysine (AP), and fish-derived gelatin (AG). Samples with varying conjugation ratios were prepared. All products formed bulk hydrogels, which were then collapsed into ultrathin membranes in a controlled environment. Membranes were evaluated for their ability to support attachment of various cell types. Cells, however, preferred the AG series of membrane. Notably, primary rabbit CEC remained attached and viable for ⩾4 weeks. The cells also stained positive for CD166, ZO-1 and Na+/K+ ATPase, indicative of function. The hydrated AG membranes allowed >96% transmittance of visible light. The membranes were typically ∼15 μm thick and did not swell significantly after immersion in PBS. Tensile strength was 49-60 MPa, while young's modulus was 525-596 MPa. This membrane thus offers great promise as a scaffold for CEC during endothelial keratoplasty.

Mini-Sheet Injection for Cultured Corneal Endothelial Transplantation.

Corneal endothelium is a single layer of hexagonal cells that maintains the corneal transparency and thickness through its barrier and pump function. For the treatment of corneal endothelial dysfunction, the transplantation of tissue-engineered corneal endothelium and direct injection of cultured corneal endothelial cells were developed because of the severe shortage of donor cornea worldwide. However, the technique difficulty or safety risk still remained. In this study, we report a novel mini-sheet injection for the cultured corneal endothelial cell transplantation and compare with the effects of single-cell injection in rabbit model. Compared with the reported single-cell injection, mini sheets promoted the adhesion and tight junction formation after injection. Rabbit corneal clarity and thickness were rapidly recovered after 7 days of mini-sheet injection, compared with 14 days of single-cell injection. Moreover, typical endothelial morphology was observed as early as 7 days after the mini-sheet injection, whereas until 21 days after the single-cell injection. These results demonstrate that the novel mini-sheet injection of corneal endothelial cells exhibited rapid adhesion, tight junction formation, and corneal clarity recovery, which may represent a more efficient method for the transplantation of cultured corneal endothelial cells.

Transplantation of Human Corneal Endothelial Cells Cultured on Bio-Engineered Collagen Vitrigel in a Rabbit Model of Corneal Endothelial Dysfunction

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.

Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Directed differentiation of human embryonic stem cells to corneal endothelial cell-like cells: A transcriptomic analysis

Corneal endothelial cells (CECs) are a monolayer of cells covering the inner-side of cornea, playing a pivotal role in keeping the cornea transparent. Because adult CECs have no proliferative capacity, the loss of CECs during aging or under pathological conditions would lead to corneal edema, eventually leading to the blindness. Clinically, donated CECs have been successfully transplanted to treat the diseases of CEC deficiency; however, the source of CEC donation is very limited. As an alternative cell source for CEC transplantation, CEC-like cells can be obtained via in vitro differentiation of human pluripotent stem cells. In this study, we introduced a modified two-stage differentiation method to convert H9 human embryonic stem cells (hESCs) to neural crest cells (NCCs), then further into CEC-like cells. The CEC-like cells treated with bovine CEC conditional medium morphologically best resembled primary CECs among all the culture conditions. By whole transcriptome analysis, we found that the typical markers of CECs, such as Na+-K+-ATPase, AQP1, Col8a and ZO-1, are highly expressed in hESC-derived CEC-like cells. By comparing RNA transcriptome of hESC-derived CEC-like cells with human primary fetal and adult CECs, we further identified shared molecular markers such as TRIT1, HSPB11, CRY1 that can be used to quality control CEC derivatives from hESCs. Our study paves the way for the quality-control and future application of hESC-derived CECs in the treatment of CEC deficiency disorders.

Stem Cell Therapy for Corneal Regeneration Medicine and Contemporary Nanomedicine for Corneal Disorders

The ocular surface is the outermost part of the visual system that faces many extrinsic or intrinsic threats, such as chemical burn, infectious pathogens, thermal injury, Stevens-Johnson syndrome, ocular pemphegoid, and other autoimmune diseases. The cornea plays an important role in conducting light into the eyes and protecting intraocular structures. Several ocular surface diseases will lead to the neovascularization or conjunctivalization of corneal epithelium, leaving opacified optical media. It is believed that some corneal limbal cells may present stem cell-like properties and are capable of regenerating corneal epithelium. Therefore, cultivation of limbal cells and reconstruction of the ocular surface with these limbal cell grafts have attracted tremendous interest in the past few years. Currently, stem cells are found to potentiate regenerative medicine by their capability of differentiation into multiple lineage cells. Among these, the most common cell sources for clinical use are embryonic, adult, and induced stem cells. Different stem cells have varied specific advantages and limitations for in vivo and in vitro expansion. Other than ocular surface diseases, culture and transplantation of corneal endothelial cells is another major issue for corneal decompensation and awaits further studies to find out comprehensive solutions dealing with nonregenerative corneal endothelium. Recently, studies of in vitro endothelium culture and ρ-associated kinase (ROCK) inhibitor have gained encouraging results. Some clinical trials have already been finished and achieved remarkable vision recovery. Finally, nanotechnology has shown great improvement in ocular drug delivery systems during the past two decades. Strategies to reconstruct the ocular surface could combine with nanoparticles to facilitate wound healing, drug delivery, and even neovascularization inhibition. In this review article, we summarized the major advances of corneal limbal stem cells, limbal stem cell deficiency, corneal endothelial cell culture/transplantation, and application of nanotechnology on ocular surface reconstruction. We also illustrated potential applications of current knowledge for the future treatment of ocular surface diseases.

Markers for Distinguishing Cultured Human Corneal Endothelial Cells from Corneal Stromal Myofibroblasts

Purpose: Eliminating contamination by corneal stromal cells is critical when preparing cultured human corneal endothelial cells (CECs) transplantation. We investigated markers for the purification of cultured human CECs and markers for excluding cultured human corneal stromal myofibroblasts (CSMFs) from cultured human CECs.Materials and methods: CECs and CSMFs were obtained from human donor corneas by culturing separately in serum-containing medium. Candidate markers of CECs and CSMFs were screened with microarray analysis in the fourth passaged CECs and CSMFs. Then, selected factors were evaluated in reverse transcription polymerase chain reaction (RT-PCR), western blot, immunocytochemistry, and flow cytometry to investigate differential markers for each cell.Results: Among the genes identified by microarray analysis, cultured human CECs, but not CSMFs, expressed integrin alpha 3 (ITGA3 and CD49c) protein according to immunocytochemistry and western blotting. Iroquois homeobox 2 (IRX2) gene was a marker that distinguished CSMFs from cultured human CECs by RT-PCR. The IRX2 gene can be used as a marker of CSMFs contaminating cultured CECs.Conclusion: These molecules could be important markers for the production of highly purified cultured CECs for regenerative medicine.

Cell-based approach for treatment of corneal endothelial dysfunction.

Decompensation of the corneal endothelium causes severe visual impairments that lead to blindness. Although corneal transplantation is a well-known effective therapy for corneal endothelial dysfunction, many patients are not afforded that therapeutic opportunity owing to the worldwide shortage of donor corneas. Thus, a tissue engineering-based therapy for treating corneal endothelial dysfunction is highly anticipated. Obstacles associated with the development of tissue engineering therapy include in vitro culture of corneal endothelial cells (CECs) and the techniques used to transplant those cells. Limited proliferation ability, cellular senescence, and fibroblastic transformation during culture are all problems associated with the cultivation of CECs. In addition, transplantation of cultured CECs is technically difficult because the corneal endothelium is composed of a fragile monolayer sheet of cells located at the posterior cornea. In this review article, we present our recent findings using a novel cell culture protocol and show that modulation of CEC adhesion properties through a Rho-kinase inhibitor enables transplantation of CECs in the form of a cell suspension without the use of a carrier. Finally, we provide an update on the clinical application status of a cell-based therapy for treating corneal endothelial dysfunction.

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells.

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.

Transplanted vascular endothelial cells to replace corneal endothelial cells by improved anterior chamber injection

To observe form and function changes of vascular endothelial cells (VEC) which were transplanted to the posterior surface of rhesus monkey cornea without Descemet's Membrane by anterior chamber injection, explore the feasibility of transplanting VEC to treat corneal endothelial injury, and find new method of corneal endothelial cell transplantation. Cultured VEC to proliferate in vitro.Rhesus monkeys were randomly divided into two groups: Experimental group (6) and control group (6) according to a random number table. The experimental group:transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet's membrane by anterior chamber injection. The control group:Tear out the Descemet's membrane by capsulotomy needle without VEC transplantation. A ultrasound apparatus was adopted to measure the postoperative thicknes of the cornea and Goldman intraocular pressure meter measuring intraocular pressure in the postoperative 7 days, 14 days, 30 days, 60 days, 90 days, organize the data and entered into the computer , applicated the software of SPSS 11.5 for data independent samples t-test and analysis of variance. The eyes were removed respectively in postoperative 30, 60, 90 days to do pathological HE dyeing and scanning electron microscopy (SEM) observation of VEC in the posterior surface of cornea graft. Corneal transpareney:In the experimental observation period (3 months), the experimental group had better transparency than the control group with normal anterior chamber depth and without bullous keratopathy. And the corneal neovascularization was exist in the cornea graft in experimental group in the third month. After 3 months, the corneal thickness of experimental group (500 ± 14) µm was significantly lower than the control group (618 ± 11) µm, Corneal thickness values between experimental and control groups were statistically significant differences in changes (all P < 0.05, t-values were -3.256, -4.419, -12.896 postoperative 1, 2, 3 months). Changes of intraocular pressure difference between the experimental group and the control was not statistically significant (all P > 0.05, t-values were -1.179, -2.166, -2.536 postoperative 1, 2, 3 months). The pathological:the cell layer was visible in the posterior surface of cornea graft. The control group:can't find the cell sample structure.SEM:Experimental group showed that VEC with irregular shape uniformly distributed on the inner surface of cornea and growing well, a small amount of white blood cells can be seen between VEC, and part of cellular debris exist in the trabecular meshwork. Control group showed a fiber material without VEC. Ultrasonic emulsification can established a repeatability and simple model of corneal endothelial injury in Rhesus monkeys.VEC can be transplanted to the corneal surface by Anterior chamber injection and the cells can grow on the surface and play a barrier role in maintaining the state of dehydration and transparency of the cornea to a certain extent. After transplantation, organizational structure and morphology of the anterior chamber angle does not produce pathological effects in the short term. Prompt that transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet's Membrane by anterior chamber injection to substitute the function of the corneal endothelial cells may be a new idea for treatment of corneal endothelial damage.

Biodegradable and Biocompatible Poly(Ethylene Glycol)‐based Hydrogel Films for the Regeneration of Corneal Endothelium

Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-μm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties.

Fabrication of a bioengineered corneal endothelial cell sheet using chitosan/polycaprolactone blend membranes

Our previous study has demonstrated cultivation of bovine corneal endothelial cells (BCECs) on the chitosan–polycaprolactone (PCL) blends. BCECs could grow well and express physiological phenotypes after PCL was introduced into chitosan by means of blending. The feasibility of using chitosan–PCL-blended membranes as scaffold and carrier for corneal endothelial cell (CEC) transplantation is worthy of more investigation. In this work, we attempt to manufacture various blended membranes to bring BCEC cultivation into harmony by hybridizing two polymers with fine adjustment. Therefore, chitosan, which does not promote BCECs maturation, and PCL, which supports, BCEC proliferation, are combined to prepare the blends. Analyses of transmittance, proliferative, abilities, phenotypic expressions, extracellular matrix (ECM) protein production, and hemotoxylin and eosin (H&E) staining were executed step-by-step. From our observations, the blended membranes united respective characteristics. The cultured BCECs on the blends illustrated normal appearance and good proliferative abilities. Immunohistochemical staining verified physiologically phenotypic expressions of ZO-1 and Na-K ATPase. Western blot analysis also confirmed the existence of collagen type IV proteins. Furthermore, the formation of a continuous monolayer of cells attached on the blended membrane was confirmed by H&E staining. These results suggested that chitosan–PCL blended membranes may be an optimized biomaterial to fabricate bioengineered corneal endothelium and show potential to facilitate CEC transplantation in the future.