Retinal Structure Measurements as Inclusion Criteria for Stem Cell–Based Therapies of Retinal Degenerations

Samuel G. Jacobson, Rodrigo Matsui, Alexander Sumaroka, and Artur V. Cideciyan

Scheie Eye Institute, Department of , Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States

Correspondence: Samuel G. Jacob- PURPOSE. We reviewed and illustrated the most optimal retinal structural measurements to son, Scheie Eye Institute, University make in stem cell clinical trials. of Pennsylvania, 51 N 39th Street, Philadelphia, PA 19104, USA; METHODS. Optical coherence tomography (OCT) and autofluorescence (AF) imaging were [email protected]. used to evaluate patients with severe visual loss from nonsyndromic and syndromic (RP), -Stargardt disease, and nonneovascular age-related macular Submitted: July 9, 2015 ABCA4 Accepted: August 21, 2015 degeneration (AMD). Outer nuclear layer (ONL), rod outer segment (ROS) layer, inner retina, ganglion cell layer (GCL), and nerve fiber layer (NFL) thicknesses were quantified. Citation: Jacobson SG, Matsui R, Su- maroka A, Cideciyan AV. Retinal RESULTS. All patients had severely reduced visual acuities. Retinitis pigmentosa patients had structure measurements as inclusion limited visual fields; maculopathy patients had central scotomas with retained peripheral criteria for stem cell–based therapies function. For the forms of RP illustrated, there was detectable albeit severely reduced ONL of retinal degenerations. Invest Oph- across the scanned retina, and normal or hyperthick GCL and NFL. Maculopathy patients had thalmol Vis Sci. 2016;57:ORSFn1– no measurable ONL centrally; it became detectable with eccentricity. Some maculopathy ORSFn9. DOI:10.1167/iovs.15-17654 patients showed unexpected GCL losses. Autofluorescence imaging illustrated central losses of RPE integrity. A hypothetical scheme to relate patient data with different phases of retinal remodeling in animal models of retinal degeneration was presented.

CONCLUSIONS. Stem cell science is advancing, but it is not too early to open the discussion of criteria for patient selection and monitoring. Available clinical tools, such as OCT and AF imaging, can provide inclusion/exclusion criteria and robust objective outcomes. Accepting that early trials may not lead to miraculous cures, we should be prepared to know why— scientifically and clinically—so we can improve subsequent trials. We also must determine if retinal remodeling is an impediment to efficacy. Keywords: retinitis pigmentosa, Usher syndrome, Stargardt disease, age-related macular degeneration

nherited retinal degenerations (IRDs) have entered an era longstanding concern to those planning stem cell therapies, also I from being incurable to having emerging therapeutic options. is addressed with illustrations of data from patients. An attempt Many previously discussed therapies are in planning stages, is made to relate the patient findings to phases of remodeling whereas others already are in early phase clinical trials.1–3 The postulated from studies of animal models.10 Details of advances expectation of investigators and patients alike is that these efforts in regenerative medicine8,11–16 are not addressed in this article, will lead to robust and safe methods with predictable outcomes but are topics covered by others in this symposium issue. that will improve vision for patients with disabling visual loss. Among current clinical trials of stem cell transplantation for retinal disease,4,5 two types of retinal degenerations are being METHODS enrolled. There are a limited number of trials in patients with forms of retinitis pigmentosa (RP), and there are more trials in Subjects patients with diseases of the macula, whether IRDs or age-related All procedures followed the Declaration of Helsinki and the macular degeneration (AMD). Different sources of stem cells are study was approved by the Institutional Review Board (IRB). being used and different delivery methods are represented.4–9 Informed consent, assent, and parental permission were The main inclusion criterion and common to all of the trials obtained, and the work was Health Insurance Portability and is, of course, seriously impaired visual function. Retinal structural Accountability Act (HIPAA)–compliant. Included were patients inclusion criteria are less well defined than functional criteria, with forms of nonsyndromic or syndromic RP (n ¼ 10; ages, 3– although many trials mention fundus photography, fluorescein 64), Stargardt disease (n ¼ 2; ages 39 and 42), and non- angiography, and optical coherence tomography (OCT). The neovascular AMD (n ¼ 2; ages 67 and 79). current study illustrates the use of segmented cross-sections of OCT to analyze retinal structural details and en face autofluo- Retinal Imaging rescence (AF) imaging to evaluate the integrity of the RPE. Patients representing the two main disease types being targeted Optical Coherence Tomography. Cross-sectional retinal for stem cell–based therapies are shown. Retinal remodeling, a images were obtained with OCT using methods previously

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described.17–21 Spectral-domain OCT (Optovue, Inc., Fremont, with nearly identical lipofusin autofluorescence on SW-RAFI CA, USA) was used to collect all data. Optical coherence (not shown), it is likely that the central macula and tomography scans were formed by laterally sampling longitu- extramacular regions correspond to severe RPE disease dinal reflectivity profiles (LRPs). High density line scan (HD possibly including complete atrophy. The question of whether line, 4091 LRPs) or line scans (average up to 32 scans, 1019 there was measurable GCL and NFL thicknesses in P1 is LRP each) 9-mm long were used to study the retina along the answered. For P1, the results are either within normal limits or horizontal and vertical meridians through the fovea; only those thickened (Figs. 1B, 1C). scans that included the foveal depression were used for Patient 2 is a 51-year-old man with a clinical diagnosis of unambiguous retinal localization. Circular scans of 3.4-mm Usher syndrome clinical subtype II (genotype unknown). The diameter centered on the optic nerve head (ONH) were ONL is measurable, but abnormally thinned; IS and OS are acquired to quantify the peripapillary retinal NFL. mostly undetectable. NIR-RAFIn shows a macular region of Optical coherence tomograms were analyzed with custom partially retained signal and the SW-RAFI results (not shown) programs (MatLab 7.5; MathWorks, Natick, MA, USA) after the are consistent with retained RPE lipofuscin. Ganglion cell layer LRPs were aligned by straightening the major RPE reflection. and NFL are within normal limits. For retinal sublaminae, signal peak assignments were based on Patient 3, a 17-year-old man with a diagnosis of early-onset our previously published work consistent with hypotheses on RP (unknown genotype), shows reduced, but detectable ONL the correspondence between OCT signals and histologically- thickness across the scan length. The laminar architecture defined layers.17,19 Specifically, the thickness of the following attributable to IS and OS is abnormally thinned. NIR-RAFIn retinal sublaminae (from vitreous to choroid) were quantified shows loss of foveal signal surrounded by detectable, but in patients and normal subjects: hyperreflective NFL, hypore- abnormal signal in the macula. The GCL and NFL are thickened flective ganglion cell layer (GCL), and hyporeflective outer relative to the normal limits (Figs. 1B, 1C). nuclear layer (ONL). Polar plots were used to display All three patients would likely satisfy inclusion criterion in a peripapillary NFL thickness. stem cell–based trial based on abnormally reduced photore- En Face AF. Imaging was performed with a confocal ceptor laminae and RPE losses that would be consistent with scanning laser ophthalmoscope (Spectralis HRA; Heidelberg the visual disturbances. As for postreceptoral inner retinal Engineering, Heidelberg, Germany) to obtain short-wavelength integrity, however, there is complexity without easy interpre- (SW-) excited reduced-illuminance autofluorescence imaging tations. Thickened GCL and/or NFL as in P1 and P3 have been (RAFI), which is based on a signal originating mostly from noted previously in forms of RP.21,24–26 The basis of such inner lipofuscin fluorophores within the RPE.22,23 In addition, near- retinopathy is not clear and it remains uncertain whether there infrared (NIR-) reflectance images and RAFI also were would be any effect of such on efficacy of treatments recorded; normalized NIR-RAFI (NIR-RAFIn) was calculated intended to replace photoreceptors. by pixel-by-pixel division of the registered images. NIR-RAFIn is thought to be based on melanin-based fluorophores within the Quantifying Retinal Laminar Integrity in RPE.23 All images were acquired with the high speed mode Maculopathies wherein a 3083308 square field was sampled onto 768 3 768 pixels. The dilated pupil of the subject was aligned with the Figure 2 illustrates cross-sectional OCT and en face NIR-RAFIn optical axis of the instrument (head position stabilized with results from patients with ABCA4-Stargardt disease (P4, P5) chin and forehead rests) in a room with dimmed ambient and with AMD (P6, P7). Patient 4 at age 39 years had a visual lights. Manufacturer’s ART feature was used whenever possible acuity of 20/250; kinetic perimetry showed that beyond a with a 21-frame average; manufacturer’s automatic normaliza- central scotoma to the V-4e target of approximately 208 in tion feature was turned off. diameter, the field to this target was within normal limits. A smaller target (I-4e) revealed a relative scotoma of approxi- mately 408 in diameter and the peripheral field was generally RESULTS reduced in extent. Optic nerve layer thickness was unmeasur- able in the foveal area but there was ONL, albeit abnormally Quantifying Retinal Laminae in Forms of reduced in thickness, eccentric to the fovea. Ganglion cell layer Nonsyndromic or Syndromic RP thickness was reduced in a relatively wide expanse of central retina but became normal at approximately 108 eccentric to Among inclusion criteria for stem cell–based trials should be the fovea. The NFL was normal or near normal. NIR-RAFIn evidence that vision loss is explained by underlying photore- showed a bilobular central macular region of demelanization. ceptor and/or RPE loss. Available modalities of retinal imaging The same pattern also was apparent on SW-RAFI (not shown) allow this basic tenet to be proved and not simply assumed. (Figs. 2A, 2B). A second Stargardt disease patient (P5, age 42) Questions also should be asked about postreceptor laminae had a of 20/320; the dimensions of the central and their integrity. Figure 1 shows cross-sectional OCT and en scotoma and peripheral field (to V-4e and I-4e targets) were face AF data from three patients with severe visual loss: very similar to those of P4. Again, the ONL was undetectable reduced visual acuity (20/250, HM, and CF, respectively for P1, centrally, but became measurable, although reduced, only at P2, and P3) and kinetic visual fields limited to a central island eccentricities of greater than approximately 78. Ganglion cell with no peripheral function. Patient 1 is a 33-year-old man with layer and NFL thicknesses, unlike in P4, were within normal Usher syndrome 3A due to a homozygous mutation (N48K/ limits. NIR-RAFIn showed a large region devoid of signal N48K) in the encoding clarin-1. In contrast to the OCT suggesting RPE atrophy; the latter was consistent with a similar from a normal subject, 1 has a very reduced, but detectable region of signal loss on SW-RAFI (not shown). ONL across the central 308. The multilaminated architecture Two patients with nonneovascular AMD (Figs. 2D, 2E) and attributable to inner (IS) and outer (OS) segments and RPE also different degrees of ONL loss across the scan length had is abnormally diminished and these layers are not clearly approximately the same visual acuity (P6, 20/320; P7, 20/250). distinguishable. Patient 6 at age 67 was symptomatic of central visual NIR-RAFIn shows a central macular region of reduced signal disturbances for approximately 2 to 3 years. She shows loss surrounded by a parafoveal ring of greater signal; extramacular of ONL and abnormal IS and OS lamination in the very central region shows further loss of NIR-RAFIn signal. Taken together retina and there are confluent drusen and drusenoid-RPE

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FIGURE 1. Cross-sectional and en face imaging in syndromic and nonsyndromic widespread retinal degenerations. (A) Upper left: OCT cross- sections across the horizontal meridian through the fovea in a normal subject and 3 patients. Outer nuclear layer is highlighted in blue and GCL in orange. Right column of images show NIR-RAFIn in the normal subject and patients. BrM/RPE, Bruch’s membrane/RPE. (B) Quantitation of ONL and GCL thickness in the patients (symbols) compared to normal results (shaded areas; mean 6 2 SD). (C) Upper: NFL thickness measurements across the horizontal meridian in patients and normal results. Lower: Polar plots of NFL thickness. N, nasal; S, superior; T, temporal; I, inferior. Inset: Location of circular scan with respect to ONH.

detachment. Outer retinal lamination becomes more normal in demelanization extending across the macula consistent with appearance at approximately 58 eccentric in the temporal SW-RAFI results retina and at approximately 78 eccentric nasally. The GCL and Again, inclusion for a central retinal stem cell–based NFL fall within normal limits. NIR-RAFIn shows a central region transplant should include a measured loss of central retinal of demelanization and SW-RAFI results are consistent with ONL that would be consistent with the visual loss; both types of patients satisfy this criterion. Despite the similarity in age, severe RPE disease or RPE atrophy. Patient 7, age 79, had 24 visual function, and diagnosis of P4 and P5, there is a definite years of symptomatic central visual disturbances and shows a reduction in the GCL in P4, but not in P5. This difference was wide area of central loss of ONL with some confluent drusen, not predictable and is complicated by the finding that NFL is and no discernible IS and OS lamination. There is thinning of generally within normal limits for both patients. Patient 7 with the GCL in temporal retina, but not in nasal retina; NFL is AMD has GCL loss in the temporal retina, but both patients within normal limits. NIR-RAFIn shows a large region of show NFL thickness within normal limits.

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FIGURE 2. Cross-sectional and en face imaging in macular degenerations. (A) Upper left: OCT cross-sections across the horizontal meridian through the fovea in two patients with Stargardt disease due to ABCA4 mutations. Highlighted layers as in Figure 1. Right column: NIR-RAFIn images. (B) Quantitation of ONL and GCL thickness in the same patients compared to normal results (shaded areas; mean 6 2 SD). (C) Upper: NFL thickness measurements across the horizontal meridian in the patients compared to normal results. Lower: Polar plots of NFL thickness. (D) Upper left: OCT cross-sections in two patients with AMD. Highlighted layers, as in Figures 1 and 2A. Right column: NIR-RAFIn images. (E) Quantitation of ONL and GCL thickness in the patients (symbols) compared with normal results (shaded areas; mean 6 2 SD). (F) Upper: NFL thickness measurements across the horizontal meridian in patients and normal results. Lower: Polar plots of NFL thickness.

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Retinal Remodeling: Phases and Surrogate Patient 9, a 39-year-old autosomal dominant (ad) RP Measures From Cross-Sectional Imaging patient with a rhodopsin mutation, shows a similar pattern of contiguous retinal regions representing different phases How the retina changes as a consequence of photoreceptor of increasing severity (Fig. 3A, right). Unlike P8, however, loss in IRDs has been a topic of scientific study for at least two P9’s expanse of normal ONL but reduced ROS (phase 1) is decades and has included histopathologic studies in human more limited and closer to the fovea. This corresponded to a donor tissue as well as in many animal models (e.g., Refs. 10, more limited visual field expanse than in P8. Other patient 27–32). A concern has been whether the many changes in scans (Figs. 3B–G) illustrate the diversity of effects in retinal inner retinal structure would alter connectivity in a way that regions with extremely reduced or nondetectable photore- would frustrate attempts to improve vision, such as could ceptors, presumed to be in remodeling phase 3. There could occur in stem cell–based transplantation strategies.31 be remarkable thickening (P10 with USH1B; P3 with early- The advent of OCT permitted us to ask in IRD patients onset RP), to somewhat thinner retina with pigment whether there was detectable change in inner retinal structure migration (hyperreflectivities blocking signals distally; ar- that could represent the effects of remodeling so well rows in P11 with adRP from RHO mutation and P12 with RP demonstrated in animals with retinal degeneration. Our many of unknown genotype) to near normal thickness of the inner studies of different forms of IRD have revealed abnormalities in retina despite loss of photoreceptors across most of the scan the inner retina associated with photoreceptor loss (e.g., Refs. length (P13 with RP and unknown genotype) to borderline 18–20, 33, 34) and more recently in Stargardt disease and or abnormally thinned retina (P14 with adRP). A sequence of AMD.35,36 Thickening of the inner nuclear layer (INL) has been progression from thickening to thinning of inner retina is notable in with photoreceptor degeneration. Thicken- speculative and awaits longitudinal data collection. If the ing of the GCL and NFL, as in some of the patients illustrated in most thinned inner retina (here illustrated with P14 data) Figures 1 and 2, also has been observed.21 proves to be a later phase of disease, it may be the most To our knowledge, there has been no attempt to date to ‘‘rewired’’10,31 and possibly the least responsive to interven- relate the phases of retinal remodeling defined in animal tion aiming to restore connectivity and visual function models with observations in human IRDs. Our attempt to do through the retina. so is presented in Figure 3. In Figure 3A (left), retinal laminar changes across a vertical OCT scan extending from the fovea to 308 in the superior retina are shown for P8, a 3-year-old DISCUSSION patient with Usher syndrome 1B due to MYO7A muta- tions.37,38 Three phases of retinal remodeling have been Quantifying the Morphologic Basis of Visual Loss 10 described in murine models of retinal degeneration. Pretreatment evaluations in the human trials of Although microscopic details are beyond the resolution of Leber congenital amaurosis due to RPE65 mutations empha- OCT, some general relationships can be drawn between the sized that retinal degenerations exist in which there is a patient data and the detailed cell biology. Due to the regional disproportionate loss of vision despite retained photoreceptors variations of severity across the retina in human RP, the (e.g., data reported previously17,39,44,45). Retinopathies with phases appear to be definable within this single scan of P8. It this complex pathophysiology at stages when there were is likely that a full study of the phases in a cohort of patients retained photoreceptors and RPE, despite severely impaired with different severities or in patients followed serially may vision, would not be appropriate candidates for stem cell– reveal the different phases as they have been described in based therapies unless other treatment strategies proved not animal models. Remodeling phase 1 is considered to occur feasible or ineffective. In modern ophthalmic clinics, the tools when there is photoreceptor stress before are available to distinguish these retinopathies from the more death. In P8, there is a wide zone of ONL that is within normal common disorders that behave as ‘‘simple photoreceptor limits in thickness—extending from the fovea to approxi- degenerations.’’ In the latter type of disorder, an OCT mately 178 into the superior retina. In this patient, photore- measurement of photoreceptor layer thickness and a colocal- ceptor stress is interpreted as reduced thickness of the rod ized measure of visual sensitivity should relate to each other outer segment (ROS) layer, that is, shortening of the ROS. according to a theoretical model based on photoreceptor Phase 2 is when there is measurable photoreceptor loss quantum catch.17,46 This is the pattern of responding that (among other complex changes10) and this is occurring in P8 occurs in most syndromic and nonsyndromic IRDs.47 In other in a zone superior to the intact ONL. There is loss of ONL and words, there should not only be measures of vision as inclusion ROS thickness, but the inner retina remains normal in criteria for stem cell–based clinical trials, but also measures of thickness. Phase 3 has been described as the occurrence of retinal structure by OCT (specifically photoreceptor structure neural remodeling, which includes further neural death, and not simply retinal thickness). If the target layer for the Muller¨ cell hypertrophy and migration of cells within the transplant is the RPE, the integrity of the RPE can be estimated distorted retina.10 In the retinal region from approximately by melanin- and lipofuscin-based AF imaging and would seem 198 to 308 in P8, there is very reduced, but detectable ONL to be a worthy measure at baseline for comparison post and no measurable ROS. The inner retina, however, has treatment.48 The observations of detectable ONL in retinal increased in thickness across the region. The detailed regions likely corresponding to RPE atrophy (made in the morphologic basis for this thickening is uncertain; we have current work) are counterintuitive, but consistent with speculated that it is due to glial cell hypertrophy. In two photoreceptor nuclei found in geographic atrophy in human murine retinal degenerations, rd16 and rhodopsin T17M eyes with AMD.49 models of CEP290-LCA and RHO-adRP, respectively, the Do we know how much retained ONL or RPE warrants reduced ONL tended to merge with the INL, which appeared consideration for best result of a specific stem cell transplant? vacuous and with larger cells, possibly from Muller¨ cell nuclei One could argue for a measurable amount of both cell and processes filling the space previously occupied by lost populations or no detectable structure, depending on results retinal cells.19,39 There are direct correlations of histopathol- of studies in animal models (e.g., see the study by MacLaren et ogy with OCT in other retinal degenerations, but the topic of al.50). Even if there is no studied answer to this question, it is remodeling was not specifically addressed (e.g., Refs. 40–43). important to know such parameters so that the field can evolve

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FIGURE 3. Surrogate measures of retinal remodeling in patients with syndromic and nonsyndromic widespread retinal degenerations. (A) Upper left and right: OCT cross-sections across the vertical meridian from the fovea into the superior retina. Outer nuclear layer and ROS are highlighted (blue and cyan, respectively) in two patients. Below the OCTs are quantitation of ONL, ROS, and inner retina in these patients compared with normal results (shaded area, mean 6 2 SD). Dashed lines across the OCT and quantitation analyses show the boundaries of the three remodeling phases. (B–G) Vertical OCT cross-sections in patients with forms of RP and very reduced ONL except at or near the fovea. Insets (upper right): Quantitation of inner retinal thickness in each patient versus normal results. Arrows in (D, E) point to hyperreflectivities from pigment migration into the inner retina.

in a more scientific and stepwise manner, and allow negative Advanced Stages of Human Retinal Degenerations and positive efficacy to be better interpreted. Currently, the May Not All Be the Same number of variables to be addressed in this field is daunting and if a human trial is deemed unsuccessful or successful by an On the topic of knowing more rather than less details of the efficacy outcome, there is a strong need to at least attempt to patients involved in stem cell–based clinical trials, despite their understand why this occurred from known details of the advanced stages of retinal degeneration, there should be an patient. attempt to determine genotypes, even if not an inclusion or

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exclusion criterion. There should be no reluctance to genotype There is the possibility that the NFL, as in some patients with these patients or at least store samples from the patients for RP, actually may be thickened but showing normal results and later genotyping. It will be argued that statistical significance of thereby ‘‘hiding’’ a thinning due to GCL losses. The asymmetry the trial outcomes would not be reached even if we do know of GCL thickness in one of the AMD patients also is somewhat genotypes, considering the small numbers of patients or single unexpected. Symptoms in this older of the two AMD patients individuals with a specific molecular cause entering early trials. were more longstanding and the extent of the central Yet, certain retinal degenerations cannot be assumed to have photoreceptor loss was greater than that in the other patient. the same postphotoreceptor retinal connectivity as others. For There was no evidence of glaucoma or other cause of GCL example, the enhanced S cone syndrome caused by NR2E3 reduction. Again, the NFL measures did not reveal notable mutations is an IRD that can masquerade as RP, but is a abnormalities. Increases in GCL and NFL thickness in forms of developmental disorder with S cones dominating the photore- RP (as well the opposite effects) have been noted previous- ceptor mosaic, few if any rods and limited numbers of other ly21,24–26 and attributed to thickened glial cell processes, cone subtypes from birth.51–55 How this genetic retinal aberrant neurites, and reactive hyperplasia of astrocytes and degeneration and its postreceptor retinal connectivity would possible vascular elements.20,28 respond to a form of stem cell therapy is uncertain,56 but it should be known that this was the treated disease; any difference between outcomes compared to other treated Acknowledgments patients would provoke a mechanism-based hypothesis to help Supported by The Ocular Research Symposia Foundation, Inc. The explain the results. Further, there is an established literature on authors alone are responsible for the content and writing of the types of syndromic and nonsyndromic forms of RP that show a paper. negative electroretinogram (ERG) suggesting prominent post- Disclosure: S.G. Jacobson,None;R. Matsui,None;A. Sumaroka, 20,57,58 receptoral dysfunction (see prior reports ) or ERGs None; A.V. Cideciyan,None suggesting an atypical retinal circuitry.59 This would be relevant disease history that could impact outcomes of a stem cell–based therapy in patients who at the time of recruitment References would not have a recordable ERG signal. 1. Jacobson SG, Cideciyan AV. Treatment possibilities for retinitis pigmentosa. N Engl J Med. 2010;363:1669–1671. The Spectre of Retinal Remodeling and Potential 2. Boye SE, Boye SL, Lewin AS, Hauswirth WW. A comprehensive Impact of Second-Order Neuron Dysfunction in review of retinal gene therapy. Mol Ther. 2013;21:509–519. Stem Cell–Based Therapies 3. Guadagni V, Novelli E, Piano I, Gargini C, Strettoi E. There is an extensive literature on the complex morphologic Pharmacological approaches to retinitis pigmentosa: a labora- modifications in postreceptoral retina after photoreceptor loss tory perspective. Prog Retin Eye Res. 2015;48:62–81. in animal models of RP (e.g., see prior reports27–32) but the 4. Nazari H, Zhang L, Zhu D, et al. Stem cell based therapies for current study is the first attempt to relate these observations age-related macular degeneration: the promises and the about phases of retinal remodeling to human inner retinal challenges. Prog Retin Eye Res. 2015;48:1–39. structural abnormalities. The human work must be extended to 5. Garcia JM, Mendon¸ca L, Brant R, et al. Stem cell therapy for determine if these inner retinal laminar changes have retinal diseases. World J Stem Cells. 2015;7:160–164. implications for efficacy in stem cell treatment trials (or any 6. Schwartz SD, Hubschman JP, Heilwell G, et al. Embryonic stem other photoreceptor-based trials). A feasible step to take is to cell trials for macular degeneration: a preliminary report. study patients at late stages of photoreceptor loss, but with Lancet. 2012;379:713–720. some retained photoreceptor laminae. Colocalized measures of visual sensitivity (rod or cone, determined by retinal location) 7. Schwartz SD, Regillo CD, Lam BL, et al. Human embryonic should be made. The same theoretical model described above stem cell-derived retinal pigment epithelium in patients with as an inclusion criterion to confirm that impaired visual loss age-related macular degeneration and Stargardt’s macular was not accompanied by disproportionate photoreceptor layer dystrophy: follow-up of two open-label phase 1/2 studies. thickness would now be used to define whether residual Lancet. 2015;385:509–516. photoreceptor structure and function are related as expected 8. Alexander P, Thomson HA, Luff AJ, Lotery AJ. Retinal pigment for a ‘‘pure photoreceptor degeneration.’’ We took the first epithelium transplantation: concepts, challenges, and future step toward this goal recently in patients with forms of ABCA4- prospects. Eye (Lond). 2015;29:992–1002. retinal degeneration.36 Regions with photoreceptor losses and 9. Song WK, Park KM, Kim HJ, et al. Treatment of macular inner retinal laminar abnormalities were compared to those degeneration using embryonic stem cell-derived retinal without inner retinal changes; colocalized rod visual function pigment epithelium: preliminary results in Asian patients. was measured in each of the areas. The model for photore- Stem Cell Reports. 2015;4:860–872. ceptor quantum catch indicated that rod visual loss in the 10. Jones BW, Marc RE. Retinal remodeling during retinal presence of inner retinal did not exceed the degeneration. Exp Eye Res. 2005;81:123–137. prediction from photoreceptor loss only. Other retinal 11. He Y, Zhang Y, Liu X, et al. Recent advances of stem cell degenerations now need to be studied similarly. therapy for retinitis pigmentosa. . 2014;15:14456– Unexpected observations concerning inner retinopathy Int J Mol Sci 14474. were made in the maculopathies illustrated in the present study (Fig. 2). These are worthy of mention considering the 12. Jayakody SA, Gonzalez-Cordero A, Ali RR, Pearson RA. Cellular stem cell trials currently listed in clinicaltrials.gov are most strategies for retinal repair by photoreceptor replacement. commonly targeting Stargardt disease and AMD patients. In one Prog Retin Eye Res. 2015;46:31–66. of the two patients with ABCA4 mutations, the central GCL 13. Wiley LA, Burnight ER, Songstad AE, et al. Patient-specific was abnormally reduced while NFL was essentially within induced pluripotent stem cells (iPSCs) for the study and normal limits. Ganglion cell layer measurements may be more a treatment of retinal degenerative diseases. Prog Retin Eye Res. direct and sensitive measure than using NFL as a surrogate.25 2015;44:15–35.

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14. Zheng A, Li Y, Tsang SH. Personalized therapeutic strategies 33. Aleman TS, Cideciyan AV, Sumaroka A, et al. Inner retinal for patients with retinitis pigmentosa. Expert Opin Biol Ther. abnormalities in X-linked retinitis pigmentosa with RPGR 2015;15:391–402. mutations. Invest Ophthalmol Vis Sci. 2007;48:4759–4765. 15. Jeon S, Oh IH. Regeneration of the retina: toward stem cell 34. Jacobson SG, Sumaroka A, Aleman TS, et al. Evidence for therapy for degenerative retinal diseases. BMB Rep. 2015;48: retinal remodelling in retinitis pigmentosa caused by PDE6B 193–199. mutation. Br J Ophthalmol. 2007;91:699–701. 16. Dang Y, Zhang C, Zhu Y. Stem cell therapies for age-related 35. Sadigh S, Cideciyan AV, Sumaroka A, et al. Abnormal macular degeneration: the past, present, and future. Clin thickening as well as thinning of the photoreceptor layer in Interv Aging. 2015;10:255–264. intermediate age-related macular degeneration. Invest Oph- . 2013;54:1603–1612. 17. Jacobson SG, Aleman TS, Cideciyan AV, et al. Identifying thalmol Vis Sci photoreceptors in blind eyes caused by RPE65 mutations: 36. Huang WC, Cideciyan AV, Roman AJ, et al. Inner and outer prerequisite for human gene therapy success. Proc Natl Acad retinal changes in retinal degenerations associated with Sci U S A. 2005;102:6177–6182. ABCA4 mutations. Invest Ophthalmol Vis Sci. 2014;55:1810– 1822. 18. Jacobson SG, Cideciyan AV, Sumaroka A, et al. Remodeling of the human retina in choroideremia: rab escort 1 (REP- 37. Jacobson SG, Aleman TS, Sumaroka A, et al. Disease 1) mutations. Invest Ophthalmol Vis Sci. 2006;47:4113–4120. boundaries in the retina of patients with Usher syndrome caused by MYO7A gene mutations. Invest Ophthalmol Vis Sci. 19. Aleman TS, Cideciyan AV, Sumaroka A, et al. Retinal laminar 2009;50:1886–1894. architecture in human retinitis pigmentosa caused by Rho- 38. Jacobson SG, Cideciyan AV, Gibbs D, et al. Retinal disease dopsin gene mutations. Invest Ophthalmol Vis Sci. 2008;49: course in Usher syndrome 1B due to MYO7A mutations. Invest 1580–1590. Ophthalmol Vis Sci. 2011;52:7924–7936. 20. Aleman TS, Soumittra N, Cideciyan AV, et al. CERKL mutations 39. Cideciyan AV, Aleman TS, Jacobson SG, et al. Centrosomal- cause an autosomal recessive cone-rod dystrophy with inner ciliary gene CEP290/NPHP6 mutations result in blindness with retinopathy. . 2009;50:5944–5954. Invest Ophthalmol Vis Sci unexpected sparing of photoreceptors and visual brain: 21. Jacobson SG, Sumaroka A, Luo X, Cideciyan AV. Retinal implications for therapy of Leber congenital amaurosis. Hum optogenetic therapies: clinical criteria for candidacy. Clin Mutat. 2007;28:1074–1083. Genet. 2013;84:175–182. 40. Huang Y, Cideciyan AV, Papastergiou GI, et al. Relation of 22. Cideciyan AV, Swider M, Aleman TS, et al. Reduced-illuminance optical coherence tomography to microanatomy in normal autofluorescence imaging in ABCA4-associated retinal degen- and rd chickens. Invest Ophthalmol Vis Sci. 1998;39:2405– erations. J Opt Soc Am A Opt Image Sci Vis. 2007;24:1457– 2416. 1467. 41. Huang Y, Cideciyan AV, Alem´an TS, et al. Optical coherence 23. Cideciyan AV, Swider M, Jacobson SG. Autofluorescence tomography (OCT) abnormalities in rhodopsin mutant trans- imaging with near-infrared excitation: normalization by genic swine with retinal degeneration. Exp Eye Res. 2000;70: reflectance to reduce signal from choroidal fluorophores. 247–251. Invest Ophthalmol Vis Sci. 2015;56:3393–3406. 42. Aleman TS, Cideciyan AV, Aguirre GK, et al. Human CRB1- 24. Walia S, Fishman GA. Retinal nerve fiber layer analysis in RP associated retinal degeneration: comparison with the rd8 patients using Fourier-domain OCT. Invest Ophthalmol Vis Crb1-mutant mouse model. Invest Ophthalmol Vis Sci. 2011; Sci. 2008;49:3525–3528. 52:6898–6910. 25. Hood DC, Lin CE, Lazow MA, Locke KG, Zhang X, Birch DG. 43. Huang WC, Wright AF, Roman AJ, et al. RPGR-associated Thickness of receptor and post-receptor retinal layers in retinal degeneration in human X-linked RP and a murine patients with retinitis pigmentosa measured with frequency- model. Invest Ophthalmol Vis Sci. 2012;53:5594–5608. domain optical coherence tomography. Invest Ophthalmol Vis 44. Jacobson SG, Cideciyan AV, Peshenko IV, et al. Determining Sci. 2009;50:2328–2336. consequences of retinal membrane guanylyl cyclase (RetGC1) 26. Xue K, Wang M, Chen J, Huang X, Xu G. Retinal nerve fiber deficiency in human Leber congenital amaurosis en route to layer analysis with scanning laser polarimetry and RTVue-OCT therapy: residual cone-photoreceptor vision correlates with in patients of retinitis pigmentosa. Ophthalmologica. 2013; biochemical properties of the mutants. Hum Mol Genet. 2013; 229:38–42. 22:168–183. 27. Li ZY, Kljavin IJ, Milam AH. Rod photoreceptor neurite 45. Jacobson SG, Cideciyan AV, Huang WC, et al. TULP1 mutations causing early-onset retinal degeneration: preserved but insen- sprouting in retinitis pigmentosa. J Neurosci. 1995;15:5429– sitive macular cones. . 2014;55: 5438. Invest Ophthalmol Vis Sci 5354–5364. 28. Milam AH, Li ZY, Fariss RN. Histopathology of the human 46. Cideciyan AV, Aleman TS, Boye SL, et al. Human gene therapy retina in retinitis pigmentosa. Prog Retin Eye Res. 1998;17: for RPE65 isomerase deficiency activates the retinoid cycle of 175–205. vision but with slow rod kinetics. Proc Natl Acad Sci U S A. 29. Banin E, Cideciyan AV, Alem´anTS,etal.Retinalrod 2008;105:15112–15117. photoreceptor-specific gene mutation perturbs cone pathway 47. Jacobson SG, Cideciyan AV, Aleman TS, et al. Usher syndromes development. Neuron. 1999;23:549–557. due to MYO7A, PCDH15, USH2A or GPR98 mutations share 30. Strettoi E, Pignatelli V. Modifications of retinal neurons in a retinal disease mechanism. Hum Mol Genet. 2008;17:2405– mouse model of retinitis pigmentosa. Proc Natl Acad Sci U S 2415. A. 2000;97:11020–11025. 48. Schmitz-Valckenberg S, Lara D, Nizari S, et al. Localisation and 31. Marc RE, Jones BW, Watt CB, Strettoi E. Neural remodeling in significance of in vivo near-infrared autofluorescent signal in retinal degeneration. Prog Retin Eye Res. 2003;22:607–655. retinal imaging. Br J Ophthalmol. 2011;95:1134–1139. 32. Jones BW, Kondo M, Terasaki H, et al. Retinal remodeling in 49. Bird AC, Phillips RL, Hageman GS. Geographic atrophy: a the Tg P347L rabbit, a large-eye model of retinal degeneration. histopathological assessment. JAMA Ophthalmol. 2014;132: J Comp Neurol. 2011;519:2713–2733. 338–345.

Downloaded from iovs.arvojournals.org on 10/01/2021 Clinical Plans for Stem Cell Therapies IOVS j Special Issue j Vol. 57 j No. 5 j ORSFn9

50. MacLaren RE, Pearson RA, MacNeil A, et al. Retinal repair by 55. Jacobson SG, Sumaroka A, Aleman TS, et al. Nuclear receptor transplantation of photoreceptor precursors. Nature. 2006; NR2E3 gene mutations distort human retinal laminar architec- 444:203–207. ture and cause an unusual degeneration. Hum Mol Genet. 51. Jacobson SG, Marmor MF, Kemp CM, Knighton RW. SWS (blue) 2004;13:1893–1902. cone hypersensitivity in a newly identified retinal degenera- 56. Strettoi E, Mears AJ, Swaroop A. Recruitment of the rod tion. Invest Ophthalmol Vis Sci. 1990;31:827–838. pathway by cones in the absence of rods. J Neurosci. 2004;24: 7576–7582. 52. Jacobson SG, Rom´an AJ, Rom´an MI, Gass JD, Parker JA. 57. Cideciyan AV, Jacobson SG. Negative electroretinograms in Relatively enhanced S cone function in the Goldmann-Favre retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1993;34: syndrome. Am J Ophthalmol. 1991;111:446–453. 3253–3263. 53. Haider NB, Jacobson SG, Cideciyan AV, et al. Mutation of a 58. Azari AA, Aleman TS, Cideciyan AV, et al. Retinal disease nuclear receptor gene, NR2E3, causes enhanced S cone expression in Bardet-Biedl syndrome-1 (BBS1) is a spectrum syndrome, a disorder of retinal cell fate. Nat Genet. 2000;24: from maculopathy to retina-wide degeneration. Invest Oph- 127–131. thalmol Vis Sci. 2006;47:5004–5010. 54. Milam AH, Rose L, Cideciyan AV, et al. The nuclear receptor 59. Aleman TS, Lam BL, Cideciyan AV, et al. Genetic heterogeneity NR2E3 plays a role in human retinal photoreceptor differen- in autosomal dominant retinitis pigmentosa with low-frequen- tiation and degeneration. Proc Natl Acad Sci U S A. 2002;99: cy damped electroretinographic wavelets. Eye (Lond). 2009; 473–478. 23:230–233.

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