Yuntao Hu

Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer

PURPOSE To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). METHODS Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). RESULTS The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. CONCLUSIONS hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.

A Novel Approach for Subretinal Implantation of Ultrathin Substrates Containing Stem Cell-Derived Retinal Pigment Epithelium Monolayer

Objective: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. Methods: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. Results: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat’s subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. Conclusion: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat’s subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

Autologous transplantation of retinal pigment epithelium-Bruch's membrane complex for hemorrhagic age-related macular degeneration.

PURPOSE To evaluate a surgical procedure for patients with hemorrhagic age-related macular degeneration (AMD). METHODS This procedure consisted of excision of the choroidal neovascular membrane and transplantation of autologous retinal pigment epithelium (RPE)-Bruchs membrane complex. The RPE-Bruchs membrane complex for transplantation was surgically developed by dissecting Bruchs membrane with the choriocapillaris from the medium size choroidal vessel layer at the midperipheral region of the choroid. Twenty-one eyes of 21 patients had this surgical procedure. Visual function tests included best corrected visual acuity (BCVA), multifocal (mf)ERG, and microperimetry. Optical coherence tomography (OCT), fluorescein angiography, and autofluorescence examinations were performed to study the status of the transplanted graft. RESULTS Among the 21 eyes, 17 with complete clinical data and qualified follow-up durations, which were 20.35 +/- 10.31 months on average, were analyzed in this series. On the last follow-up visit, the mean for the ETDRS scores increased from 28.65 +/- 23.99 before surgery to 47.76 +/- 17.22 after surgery. Microperimetry showed that after surgery, seven eyes gained central fixation at the 12-month follow-up examination. However, two eyes lost their central fixation on the last follow-up visit. Fourteen (82.35%) of the transplanted patches preserved normal color without depigmentation. Among the 21 eyes, proliferative vitreoretinopathy (PVR) occurred in 3 (14.29%), and a recurrent neovascular membrane was observed in one eye (4.76%). CONCLUSIONS The transplantation of the autologous RPE-Bruchs membrane complex can increase the visual acuity of patients with hemorrhagic AMD. The surviving transplanted graft with functional overlying retina was observed after surgery.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

PURPOSE To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. RESULTS Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Submacular DL-α-Aminoadipic Acid Eradicates Primate Photoreceptors but Does Not Affect Luteal Pigment or the Retinal Vasculature

PURPOSE Macular telangiectasia type 2 (MT2) is a condition of uncertain etiology characterized by retinal vascular abnormalities, depletion of luteal pigment, and photoreceptor loss. To model this condition, the authors recently used a purportedly glial-selective toxin, DL-α-aminoadipic acid (DL-α-AAA), to test the effect of Müller cell disruption on the blood-retinal barrier in rats. In this study, they investigated macular changes after subretinal injection of DL-α-AAA in monkeys. METHODS Various doses of DL-α-AAA were injected beneath the macula in eight monkey eyes. Eyes were examined by multifocal electroretinography (mfERG), optical coherence tomography (OCT), fundus autofluorescence, color photography, and fluorescein angiography. Five months after injection, eyes were examined by histology and immunohistochemistry for changes in photoreceptors and the retinal glia. In vitro studies evaluated the effect of DL-α-AAA on 661W cone photoreceptor viability. RESULTS Subretinal injection of DL-α-AAA resulted in virtually complete ablation of photoreceptors in the injected area, as shown by OCT and histology, and severely impaired mfERG responses. Müller cells, albeit activated, survived the injury. Macular pigment remained unchanged in the central fovea. Subretinal injection of DL-α-AAA did not induce vascular leakage, though it increased the fundus autofluorescence. DL-α-AAA had a dose-dependent toxic effect on 661W photoreceptors. CONCLUSIONS Submacular injection of DL-α-AAA induced severe damage to photoreceptors but failed to eliminate Müller cells in monkeys. Central macular pigment persisted despite loss of photoreceptors, and the retinal vasculature was unaffected. These observations may have significance in studying the roles of different cellular components in the pathogenesis of MT2.

Photoreceptors Repair by Autologous Transplantation of Retinal Pigment Epithelium and Partial-Thickness Choroid Graft in Rabbits

PURPOSE To investigate whether autologous retinal pigment epithelium (RPE) and a partial-thickness graft can repair degenerated photoreceptors overlying a mechanically damaged Bruchs membrane. METHODS Twenty-one pigmented rabbits were used in the study. Abrasive debridement of the RPE was performed with a metal cannula after superior retinal bleb detachment in 20 rabbits. The graft was prepared beneath the inferior retina and was transplanted to the debridement area 14 days later. Debridement-only sites served as the control. Tissue sections were evaluated by light microscopy and transmission electron microscopy at 7 days, 1 month, and 3 months after transplantation, corresponding to 21 days, 45 days, and 3 months after debridement, respectively. RESULTS When analyzed at 7 days after transplantation, short buds of inner segment with regularly organized outer nuclear layer were observed. The outer segments (OS) were of insufficient length to be observed, but by 1 and 3 months, a significant elongation of the OS was detected. In control retinas from 21 days (corresponding to 7 days after transplantation) to 3 months after RPE debridement, the outer nuclear layer cells were disorganized and diminished. CONCLUSIONS This study showed that autologous RPE and partial-thickness choroid graft have the capacity not only to support photoreceptor cell survival, but also to initiate early repair mechanisms, as exhibited by outer segment regeneration.

Risk factors, anatomical, and visual outcomes of injured eyes with proliferative vitreoretinopathy: eye injury vitrectomy study.

Purpose: To investigate potential risk factors for development of proliferative vitreoretinopathy (PVR) post trauma and evaluate the effect of PVR on anatomical and visual outcomes in injured eyes. Methods: Overall, 179 eyes with PVR and 221 eyes without PVR after injury were selected from the database of the Eye Injury Vitrectomy Study, a multicenter cohort study launched in 1997. Multivariate logistic regression was used to ascertain the independent risk factors for development of PVR and to evaluate the influence of PVR on anatomical and visual outcomes. Results: An interval of injury and vitrectomy of more than 28 days (odds ratio, 139.25; confidence interval, 50.09–387.10), severe vitreous hemorrhage (odds ratio, 2.72; confidence interval, 1.13–6.52), and total retinal detachment (odds ratio, 12.67; confidence interval, 3.96–40.52) were important independent risk factors for PVR. One hundred and fifteen eyes (52.0%) and 49 eyes (27.4%) without and with PVR, respectively, were anatomically restored with ambulant visual acuity (≥4/200). Proliferative vitreoretinopathy, poor initial visual acuity, relative afferent pupillary defect, total retinal detachment, and retinal tear or retinal defect were unfavorable prognostic indicators. Conclusion: Proliferative vitreoretinopathy occurs frequently in injured eyes and is associated with poor outcomes. Its onset depends on interval of injury and vitrectomy, wound location, vitreous hemorrhage, and retinal detachment. Early vitrectomy (before 2 weeks) and aggressive therapy should be considered for specific high-risk cases.

Manganese‐enhanced MRI optic nerve tracking: effect of intravitreal manganese dose on retinal toxicity

The aim of this study was to provide data on the dose dependence of manganese‐enhanced MRI (MEMRI) in the visual pathway of experimental rats and to study the toxicity of MnCl2 to the retina. Sprague–Dawley rats were intravitreally injected with 2 μL of 0, 10, 25, 50, 75, 100, 150 and 300 mm MnCl2, respectively. The contrast‐to‐noise ratio (CNR) of MEMRI for optic nerve enhancement was measured at different concentrations of MnCl2. Simultaneously, the toxicity of manganese was evaluated by counting retinal ganglion cells and by retinal histological examination using light microscopy and transmission electron microscopy. The CNR increased with increasing concentration of MnCl2 up to 75 mm. Retinal ganglion cell densities were reduced significantly when the concentration of MnCl2 in the intravitreal injection was equal to or greater than 75 mm. Increasing numbers of ribosomes in retinal ganglion cells were first detected at 25 mm of MnCl2. The retinal toxicity of MnCl2 at higher concentration also included mitochondrial pathology and cell disruption of retinal ganglion cells, as well as abnormalities of photoreceptor and retinal pigment epithelium cells. It can be concluded that intravitreal injection of MnCl2 induces retinal cell damage that appears to start from 25 mm. The concentration of MnCl2 should not exceed 25 mm through intravitreal injection for visual pathway MEMRI in the rat. Copyright

Autologous Transplantation of RPE with Partial- Thickness Choroid after Mechanical Debridement of Bruch Membrane in the Rabbit

PURPOSE An improved translocation technique for autologous retinal pigment epithelium (RPE) transplantation is presented. The graft consists of a sheet of a partial-thickness choroid with RPE attached. METHODS Twenty-seven pigmented rabbits were used in this study. After mechanical debridement of Bruch membrane, partial-thickness RPE-choroid sheets were transplanted onto the subretinal space in 25 rabbits. The animals were examined by fundus photographs and fluorescein angiographs and were killed postoperatively at 1, 2, 4, 12, and 24 weeks. Eyecups containing the grafts were examined by light microscopy and immunohistochemistry. In addition, two partial-thickness RPE-choroid sheets were analyzed by transmission electron microscopy (TEM). RESULTS TEM revealed that the partial-thickness RPE-choroid graft consisted of retinal pigment epithelial cells, Bruch membrane, choriocapillaris, and ruptured middle vessels. The thickness of the graft was approximately 50 to 60 microm. Fluorescein angiography revealed neither fluorescein leakage nor staining in the graft at early or late phase. Light microscopy revealed that in 17 experiments in which the graft survived, the neural retina remained intact; however, in eight experiments with unsuccessful grafts, the neural retina degenerated. The surviving graft showed revascularization and monolayered retinal pigment epithelial cells. Furthermore, in sections in which the neural retina over the graft remained intact, all retinal pigment epithelial cells in the graft and rhodopsin in photoreceptor outer segments were positively labeled with anticellular retinaldehyde-binding protein antibodies and anti-opsin antibodies, respectively. CONCLUSIONS A partial-thickness RPE-choroid graft showed improved integration with the host choroid and photoreceptors. This technique has the potential to be a treatment for age-related macular degeneration.

In vivo detection of hESC-RPE cells via confocal near-infrared fundus reflectance.

BACKGROUND AND OBJECTIVE To investigate whether the confocal near-infrared reflectance (NIR) imaging modality could detect the in vivo presence of retinal pigment epithelium cells derived from embryonic human stem cells (hESC-RPE) implanted into the subretinal space of the Royal College of Surgeons (RCS) rat. MATERIALS AND METHODS Monthly NIR images were obtained from RCS rats implanted with either hESC-RPE seeded on a parylene membrane (n = 14) or parylene membrane without cells (n = 14). Two independent, masked investigators graded the images. Histology and immunohistochemistry were performed at different time points (150, 210, and 270 postnatal days of age). RESULTS NIR images revealed that an average of 20.53% of the parylene membrane area was covered by hESC-RPE. RPE-65 and TRA-1-85 confirmed the presence of human-specific RPE cells in those animals. No areas corresponding to cells were found in the group implanted with membrane only. Intergrader agreement was high (r = 0.89-0.92). CONCLUSION The NIR mode was suitable to detect the presence of hESC-RPE seeded on a membrane and implanted into the subretinal space of the RCS rat.