Junichi Kiryu

Characterization of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Sheets Aiming for Clinical Application

Summary Age-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD.

Reduced retinal ganglion cell complex thickness in patients with posterior cerebral artery infarction detected using spectral-domain optical coherence tomography

PurposeTo report a reduction in macular ganglion cell complex (GCC) thickness detected with spectral-domain optical coherence tomography (SD-OCT) in patients with homonymous hemianopia following acquired cerebral damage.MethodsWe analyzed case reports of three patients with unilateral posterior cerebral artery (PCA) infarction. Three patients (aged 66, 68, and 71xa0years) with left homonymous hemianopia due to infarction of the right PCA territory were studied using SD-OCT. The duration of the lesions from the onset ranged from 12 to 36xa0months.ResultsIn all of these patients, although optic atrophy and retinal nerve fiber layer defects were not detected on funduscopy, GCC thinning was demonstrated in the hemiretinae corresponding to the affected hemifields. Macular GCC measurements showed the localized defects of retinal ganglion cells (RGCs) more clearly than circumpapillary retinal nerve fiber layer measurements. Total and outer retinal thicknesses were not found to be significantly affected.ConclusionOur findings suggest that degeneration of RGCs can occur within a few years after PCA infarction.

Sectoral analysis of the retinal nerve fiber layer thinning and its association with visual field loss in homonymous hemianopia caused by post-geniculate lesions using spectral-domain optical coherence tomography

PurposeTo report a sectoral analysis of circumpapillary retinal nerve fiber layer (cpRNFL) thinning and its association with visual field loss using spectral-domain optical coherence tomography (SD-OCT) in patients with homonymous hemianopia following acquired post-geniculate visual pathway damage.Patients and methodsSeven patients with homonymous hemianopia due to unilateral acquired post-geniculate visual pathway lesions were studied. The average duration from the onset of brain lesions to the initial visit was 49.8xa0months. Forty-nine normal control subjects without visual field defects, as confirmed using a Humphrey visual field analyzer, were also enrolled. Measurement of the cpRNFL thickness was performed at the initial visit and 24xa0months using SD-OCT (RTVue-100® OCT). The cpRNFL thickness was divided into eight sectors (superior temporal: ST, temporal upper: TU, temporal lower: TI, inferior temporal: IT, inferior nasal: IN, nasal lower: NL, nasal upper: NU, superior nasal: SN). The eye on the same side asxa0the occipital lobe lesions was defined as the ipsilateral eye, and the eye on the opposite side was defined as the contralateral eye.ResultsThe average cpRNFL thickness in the homonymous hemianopic eyes was significantly reduced as compared with that seen in the normal controls, except for the ipsilateral eyes at the initial visit. Four of the eight sectors of the cpRNFL thickness in the homonymous hemianopic eyes were significantly reduced compared with that noted in the normal controls. In the ipsilateral eyes, the cpRNFL thickness in the ST, TU, TL, and IT sectors was significantly reduced at both the initial visit and 24xa0months. In the contralateral eyes, the cpRNFL thickness in the TU, TL, IT, and SN sectors was significantly reduced at both the initial visit and 24xa0months. The reduction of the quadrantic cpRNFL thickness significantly correlated with some of the visual field parameters, in accordance with the structure–function relationship. In the contralateral eyes, the T and I quadrant cpRNFL thickness correlated with the mean deviation and hemianopic field total deviation at 24xa0months. In the ipsilateral eyes, the S, T, and I quadrant cpRNFL thickness correlated with mean deviation. However, there were no correlations between the cpRNFL thickness and visual field parameters at the initial visit.ConclusionsA reduction of the cpRNFL thickness corresponding to the hemianopic visual field loss due to acquired post-geniculate visual pathway lesions was detected using SD-OCT, and the change was more evident at 24xa0months than at the initial visit. The latter finding suggests that this change is, at least partially, caused by transsynaptic retrograde degeneration.

Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE.

PURPOSEnFor the transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), determination of the maturation status of these cells is essential, and the degree of pigmentation (dPG) can serve as a good indicator of this status. The aim of this study was to establish a method of objectively and quantitatively evaluating the dPG of hiPSC-RPE.nnnMETHODSnTwo observers determined the dPG subjectively by observing recorded images of hiPSC-RPE as follows: the dPG of a single cell was classified into three different pigmentation stages, and the overall dPG was compared between two cell groups to identify the group with the higher dPG. The κ statistic was applied to assess interobserver reproducibility. Next, the dPG of single cells and cell groups was objectively determined by the lightness of the hue, saturation, and value (HSL) color space, and the correlation between the subjective evaluation and time-dependent change in the objective dPG of hiPSC-RPE was investigated.nnnRESULTSnThe κ statistic was 0.88 and 0.81 in the single-cell and cell-group observations, respectively. The objective dPG of single cells and cell groups was highly correlated with the subjective dPG. However, the observers were occasionally unable to subjectively determine the group with the higher dPG. The objective dPG increased in a time-dependent manner.nnnCONCLUSIONSnThe lightness of the HSL color space can be used to objectively and quantitatively evaluate the dPG of hiPSC-RPE in culture. The objective evaluation was consistent and was able to better identify small differences than subjective evaluation.

Factors affecting imaging of spectral-domain optical coherence tomography in gas-filled eyes after macular-hole surgery

PurposeWe investigated factors affecting imaging of spectral-domain optical coherence tomography (SD-OCT) in gas-filled eyes after macular-hole (MH) surgery.MethodsThe study comprised 25 eyes in 23 patients, eight men and 15 women, with a mean age of 66.9xa0(48–77)xa0years, all of whom had undergone vitrectomy for MH. Sulfur hexafluoride (SF6) gas was used as a tamponade, and the patients were maintained in a prone position after surgery. The SD-OCT imaging was performed on postoperative days 1 and 2.ResultsThe success rate of imaging was 88.0% (22/25 eyes) on postoperative day 1 and 95.8% (23/24 eyes) on postoperative day 2. The rate of MH closure in successful imaging cases was 81.8% (18 eyes) on postoperative day 1 and 95.7% (22 eyes) on postoperative day 2. The imaging accuracy in gas-filled eyes increased by setting the focus value of the OCT to a myopic refractive shift.ConclusionThe imaging of many gas-filled eyes following MH surgery is possible, even at the early postoperative stage, by setting the focus value of the OCT to a myopic refractive shift.

Time Course of Macular and Peripapillary Inner Retinal Thickness in Non-arteritic Anterior Ischaemic Optic Neuropathy Using Spectral-Domain Optical Coherence Tomography

ABSTRACT To report a time course of the ganglion cell complex (GCC) and circumpapillary retinal nerve fibre layer (cpRNFL) thicknesses using spectral-domain optical coherence tomography in patients with non-arteritic anterior ischaemic optic neuropathy (NAION), five patients with unilateral NAION were studied (the average age of 66.8 ± 7.8 years old). Forty-one age-matched normal controls were also enrolled. The GCC and cpRNFL thicknesses were measured at the initial visit and at 1, 3, 6, and 12 months using RTVue-100. The GCC thickness and the cpRNFL thickness of the patients were compared with those of the normal controls. The GCC thickness in the NAION patients was 96.49 μm at the initial visit, 84.28 μm at 1 month, 74.26 μm at 3 months, 71.23 μm at 6 months, and 69.51 μm at 12 months. The values at 1, 3, 6, and 12 months were significantly reduced (p < 0.01). The cpRNFL thickness at the initial visit was significantly increased, whereas the values at 6 and 12 months were significantly reduced (p < 0.01). The GCC thickness is more useful for the detection of retinal ganglion cell loss at an early stage than the cpRNFL thickness, because the GCC thickness is unaffected by optic disc swelling at the initial visit, unlike the cpRNFL thickness.

Evaluation of the Surgical Device and Procedure for Extracellular Matrix–Scaffold–Supported Human iPSC–Derived Retinal Pigment Epithelium Cell Sheet Transplantation

PurposenTo develop a clinically applicable transplantation device and surgical procedure for extracellular matrix-scaffold-supported human-induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cell sheet transplantation for clinical use.nnnMethodsnThe developed surgical device consisted of a custom-designed hand piece and a cannula. The subretinal transplantation of hiPSC-RPE cell sheets was performed in 12 rabbits. The results evaluated were the graft condition (damage or fold), side (front or back), position (center, near, far), and direction (anterior, posterior, right, left) immediately after surgery and the graft condition (shrinking or fold) 2 weeks after surgery. These results were evaluated by fundus photography and optical coherence tomography, followed by immersion-fixed histology.nnnResultsnAll grafts could be transplanted without obvious damage. The transplanted grafts included 2 of 12 folded grafts, 12 of 12 front side, 12 of 12 center position, 10 of 12 anterior direction, and 2 of 12 right direction immediately after surgery, whereas transplantation with a distance between an inlet and an outlet greater than graft and the coaxial direction of the flow paths and the insertion device posed the correct condition and direction. Two weeks after the surgery, the transplanted grafts included two folded grafts and four shrunken grafts; however, complete drainage of subretinal fluid for adhesion between the graft and the host prevented shrunken grafts.nnnConclusionsnA developed surgical device and procedure allow grafts to be transplanted into the targeted transplantation site safely and reproducibly. This surgical method will provide additional information on the advancement of future RPE transplantation therapies.

Experimental proliferative vitreoretinopathy in rabbits by delivery of bioactive proteins with gelatin microspheres

Graphical abstract Figure. No caption available. &NA; Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon‐beta (IFN&bgr;). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFN&bgr; solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF‐impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFN&bgr;‐impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFN&bgr;, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFN&bgr;‐impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres.

Macular retinal and choroidal thickness in unilateral amblyopia using swept-source optical coherence tomography

BackgroundTo investigate macular retinal and choroidal thickness in amblyopic eyes compared to that in fellow and normal eyes using swept-source optical coherence tomography (SS-OCT).MethodsThis study examined 31 patients with hyperopic anisometropic amblyopia (6.9 ± 3.8 years, mean ± standard deviation), 15 patients with strabismic amblyopia without anisometropia (7.9 ± 4.2 years), and 24 age-matched controls (7.8 ± 3.3 years). Retinal and choroidal thickness was measured by 3D scans using SS-OCT. A 6-mm area around the fovea was automatically analyzed using the Early Treatment Diabetic Retinopathy Study map. The thickness from SS-OCT was corrected for magnification error using individual axial length, spherical refraction, cylinder refraction, and corneal radius. Retinal thickness was divided into the macular retinal nerve fiber layer (mRNFL), ganglion cell layer + inner plexiform layer (GCL+IPL), ganglion cell complex (GCC), and the inner limiting membrane to the retinal pigment epithelium (ILM-RPE) thickness. Retinal and choroidal thickness was compared among amblyopic, fellow, and normal eyes.ResultsIn both amblyopia groups, there was no significant difference in the mRNFL, GCL+IPL, and GCC thicknesses among the amblyopic, fellow, and control eyes. In the anisometropic amblyopia group, choroidal thickness (subfovea, center 1 mm, nasal and inferior of the inner ring, nasal of the outer ring, and center 6 mm) of amblyopic eyes were significantly greater than that of fellow and normal eyes. In contrast, none of the choroidal thicknesses were significantly different among the investigated eyes in the strabismic amblyopia group.ConclusionsWe found no significant difference in inner retinal thickness in patients with unilateral amblyopia. Although there were significant differences in choroidal thickness with hyperopic anisometropic amblyopia, there was no significant difference for the strabismic amblyopia. The discrepancy in choroidal thickness between the two types of amblyopia may be due to both differences in ocular size and underlying mechanism.

Intravitreal Recombinant Tissue Plasminogen Activator Without Gas Injection in a Patient with Massive Submacular Hemorrhage Associated with Age-Related Macular Degeneration: A Case Report

Introduction: Submacular hemorrhage (SMH) is a leading cause of severe visual loss. Pneumatic displacement of SMH from the macular area using intravitreal injection of tissue plasminogen activator (tPA) and gas and vitrectomy with subretinal injection of tPA and intravitreal injection of gas have recently been used as the standard therapies for SMH patients. However, little has been reported on single intravitreal administration of tPA for SMH patients. Case report: A 62-year-old male patient noted both blurred vision in his left eye and difficulty speaking (dysarthria) 1 day before admission. He was diagnosed with both massive SMH and cerebral infarction. Funduscopy revealed that the fovea was shifted to the inferonasal side due to a massive subretinal blood clot, and optical coherence tomography (OCT) revealed steep retinal detachment, hyper-reflective material representing a blood clot under the retina, and multiple large retinal pigment epithelial detachments (PEDs). The patient received a single intravitreal administration of tissue plasminogen activator. After vitreous injection, nearly all of the massive subretinal blood clot moved to the peripheral retina, and the fovea returned to the appropriate position. His best corrected visual acuity improved from 20/250 to 20/100. Conclusion: The present study showed a favorable outcome of a patient with a massive SMH complicated by cerebral infarction after receiving a single intravitreal administration of tPA. We hope that the single intravitreal administration of tPA can be applied for SMH patients who are not appropriate for surgery.