Proinflammatory Pathways Are Activated in the Human Q344X Rhodopsin Knock-In Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Rod nuclear architecture determines contrast transmission of the retina and behavioral sensitivity in mice.

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and the underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture at the expense of image detail. Here, we show that retinal optical quality improves 2-fold during terminal development, and that this enhancement is caused by nuclear inversion. We further demonstrate that improved retinal contrast transmission, rather than photon-budget or resolution, enhances scotopic contrast sensitivity by 18-27%, and improves motion detection capabilities up to 10-fold in dim environments. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast transmission as a decisive determinant of mammalian visual perception.

Visual Contrast Sensitivity Correlates to the Retinal Degeneration in Rhodopsin Knockout Mice.

Purpose: Clinical manifestations of photoreceptor degeneration include gradual thinning of the outer nuclear layer (ONL) and progressive reduction of electroretinogram (ERG) amplitudes and vision loss. Although preclinical evaluations of treatment strategies greatly depend on rodent models, the courses of these changes in mice remain unclear. We thus sought to investigate the temporal correlations in changes of spatial vision, ERG response, and ONL thickness in mice with progressive photoreceptor degeneration. Methods: Adult wild-type (WT) mice and mice carrying rhodopsin deficiency (Rho-/-), a frequently used mouse model of human retinitis pigmentosa, were selected for investigation. Mouse spatial vision, including visual acuity (VA) and contrast sensitivity (CS), was determined using optomotor response (OMR) assays; ONL thickness was quantified by spectral-domain optical coherence tomography (SD-OCT), and ERG was performed to evaluate retinal functions. The mice were killed when they were 14 weeks old, and the cone photoreceptors in retinal sections were counted. Results: Spatial vision, ONL thickness, and ERG amplitudes remained stable in WT mice at all examined time points. While 6-week-old Rho-/- mice had VA, CS, as well as ERG responses similar to those of WT mice, progressive reductions in the spatial vision and retinal functions were recorded thereafter. Most tested 12-week-old Rho-/- mice had no visual-evoked OMR and ERG responses. Moreover, CS, but not VA, displayed a linear decline that was closely associated with ONL thinning, reduction of ERG amplitudes, and loss of cones. Conclusions: We presented a comprehensive study of the relation between the changes of spatial vision, retinal function, and ONL thickness in postnatal week (PW)6 to PW12 Rho-/- mice. CS is a more sensitive indicator of spatial vision compared to VA, although both are required as separate parameters for monitoring the visual changes in retina undergoing photoreceptor degeneration.

Photoreceptor preservation induced by intravitreal controlled delivery of GDNF and GDNF/melatonin in rhodopsin knockout mice.

Purpose: To evaluate the potential of a poly(lactic-co-glycolic acid) (PLGA)-based slow release formulation of glial cell line-derived neurotrophic factor (GDNF) alone or in combination with melatonin to rescue photoreceptors in a mouse model of retinal degeneration. Methods: GDNF and GDNF/melatonin-loaded PLGA microspheres (MSs) were prepared using a solid-in-oil-in-water emulsion solvent extraction-evaporation technique. A combination of PLGA and vitamin E (VitE) was used to create the microcarriers. The structure, particle size, encapsulation efficiency, and in vitro release profile of the microparticulate formulations were characterized. Microparticulate systems (non-loaded, GDNF, and GDNF/melatonin-loaded MSs) were administered intravitreally to 3-week-old rhodopsin knockout mice (rho (-/-); n=7). The functional neuroprotective effect was assessed with electroretinography at 6, 9, and 12 weeks old. The rescue of the structure was determined with photoreceptor quantification at 12 weeks (9 weeks after administration of MSs). Immunohistochemistry for photoreceptor, glial, and proliferative markers was also performed. Results: The microspheres were able to deliver GDNF or to codeliver GDNF and melatonin in a sustained manner. Intravitreal injection of GDNF or GDNF/melatonin-loaded MSs led to partial functional and structural rescue of photoreceptors compared to blank microspheres or vehicle. No significant intraocular inflammatory reaction was observed after intravitreal injection of the microspheres. Conclusions: A single intravitreal injection of GDNF or GDNF/melatonin-loaded microspheres in the PLGA/VitE combination promoted the rescue of the photoreceptors in rho (-/-) mice. These intraocular drug delivery systems enable the efficient codelivery of therapeutically active substances for the treatment of retinal diseases.

Müller Cell Biological Processes Associated with Leukemia Inhibitory Factor Expression.

Müller cells provide support to photoreceptors under many conditions of stress and degeneration. Leukemia inhibitory factor is known to be expressed in Müller cells, which is necessary to promote photoreceptor survival in stress. We hypothesize that Müller cells that express LIF are undergoing other biological processes or functions which may benefit photoreceptors in disease. In this study, we analyze an existing single Müller cell microarray dataset to determine which processes are upregulated in Müller cells that express LIF, by correlating LIF expression to the expression of other genes using a robust correlation method. Some enriched processes include divalent inorganic cation homeostasis, negative regulation of stem cell proliferation, and gamma-glutamyl transferase activity.

Electrophysiology Alterations in Primary Visual Cortex Neurons of Retinal Degeneration (S334ter-line-3) Rats.

The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration.

Guanylate cyclase 1 relies on rhodopsin for intracellular stability and ciliary trafficking.

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin's role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein.

Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype.

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.

Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependent damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.

Explant cultures of Rpe65-/- mouse retina: a model to investigate cone opsin trafficking.

PURPOSE: In the absence of 11-cis retinal (e.g., Rpe65⁻/⁻), the chromophore for all pigments, cone opsins are mislocalized in vivo. Using the systemic application of 11-cis retinal, appropriate protein localization can be promoted. Here, we asked whether explant cultures of Rpe65⁻/⁻ mouse retina are amenable to screening retinoids for their ability to promote opsin trafficking. METHODS: Retina-retinal pigment epithelium (RPE) cultures were prepared from 7-day-old Rpe65⁻/⁻ Rho⁻/⁻ or wild-type pups and cultured for 11 days. Explants were treated with retinoids throughout this period. Ultraviolet (UV)-opsin trafficking was analyzed by immunohistochemistry and quantitative image analysis, while its messenger RNA expression was examined by quantitative real-time PCR, and the interaction of retinoids with UV-opsin was probed in transducing-activation assays. RESULTS: In wild-type explant cultures, UV-opsin was restricted to the outer segments, whereas in those derived from Rpe65⁻/⁻ Rho⁻/⁻ mice, opsin trafficking was impaired. In Rpe65⁻/⁻ Rho⁻/⁻ explants, administration of 11-cis retinal, 11-cis retinol or retinoic acid (RA) reversed the opsin trafficking phenotype. RA analogs designed to act by binding to the retinoic acid receptor or the retinoid X-receptor, however, had no effect. RA was shown to interact with the UV-cone opsin, demonstrated by its ability to effect ligand-dependent activation of transducin by UV-cone opsin. All compounds tested increased cone opsin messenger RNA expression. CONCLUSIONS: Cone-opsin trafficking defects were replicated in Rpe65⁻/⁻ Rho⁻/⁻ retina-RPE cultures, and were reversed by 11-cis retinal treatment. Comparing the effects of different retinoids on their ability to promote UV-opsin trafficking to outer segments confirmed the critical role of agents that bind in the retinoid binding pocket. Retinoids that act as transcription factors, however, were ineffective. Thus, organ cultures may be a powerful low-throughput screening tool to identify novel compounds to promote cone survival.

Reprogramming of adult rod photoreceptors prevents retinal degeneration.

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Gene expression changes within Müller glial cells in retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. In this article, we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knockout (Rhod-ko) models. The RNA repertoire of single MGCs was comprehensively profiled, and a comparison was made between MGCs from wild-type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death. METHODS: Retinas were dissociated, and single MGCs were chosen under a dissecting microscope using a micropipette. Single cell cDNAs were generated and genome-wide profiles were obtained by hybridization to Affymetrix arrays. A comparison was made among all samples to discover the changes in gene expression during the periods of rod and cone photoreceptor death. RESULTS: MGCs respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of glial fibrillary acidic protein (Gfap). Many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune-related response. CONCLUSIONS: A high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells and/or inherent heterogeneity among MGCs.

Feedback from rhodopsin controls rhodopsin exclusion in Drosophila photoreceptors.

Sensory systems with high discriminatory power use neurons that express only one of several alternative sensory receptor proteins. This exclusive receptor gene expression restricts the sensitivity spectrum of neurons and is coordinated with the choice of their synaptic targets. However, little is known about how it is maintained throughout the life of a neuron. Here we show that the green-light sensing receptor rhodopsin 6 (Rh6) acts to exclude an alternative blue-sensitive rhodopsin 5 (Rh5) from a subset of Drosophila R8 photoreceptor neurons. Loss of Rh6 leads to a gradual expansion of Rh5 expression into all R8 photoreceptors of the ageing adult retina. The Rh6 feedback signal results in repression of the rh5 promoter and can be mimicked by other Drosophila rhodopsins; it is partly dependent on activation of rhodopsin by light, and relies on G(αq) activity, but not on the subsequent steps of the phototransduction cascade. Our observations reveal a thus far unappreciated spectral plasticity of R8 photoreceptors, and identify rhodopsin feedback as an exclusion mechanism.

Combining chondroitinase ABC and growth factors promotes the integration of murine retinal progenitor cells transplanted into Rho(-/-) mice.

PURPOSE: The aim of this study is to investigate the synergistic effect of chondroitinase ABC and growth factors in the integration of murine retinal progenitor cells (mRPCs) transplanted into Rho(-/-) mice. METHODS: mRPCs from P1 green fluorescent protein-transgenic mice were isolated and expanded for transplantation. All mRPCs of 20 passages or less were transplanted into the subretinal space of B6 mice together with chondroitinase ABC, and into Rho(-/-) mice combined with chondroitinase ABC, N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), and insulin growth factor (IGF)-1. Cell counts were used to examine the migration and survival rate of mRPCs in B6 mice. Immunohistochemistry was used to evaluate the differentiation and integration of mRPCs in B6 and Rho(-/-) mice. RESULTS: Our results show that substantial numbers of mRPCs migrated and survived in the retina when transplanted with chondroitinase ABC into B6 and Rho(-/-) mice. Chondroitinase ABC disrupted the glial scar around the mRPCs in the subretinal space. Only a few mRPCs expressed recoverin in B6 mice. More mRPCs expressed rhodopsin, recoverin, and synaptophysin after transplantation into Rho(-/-) mice when combined with chondroitinase ABC and growth factors. CONCLUSIONS: The synergistic effect of chondroitinase ABC and growth factors facilitates the anatomic integration of mRPCs transplanted into Rho(-/-) mice.

Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor-specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones.

Optical intrinsic signal mapping of rod- and cone-mediated visual cortex responses in mice.

We used optical imaging of intrinsic signals to study visual cortex responses in three mouse strains: wild-type (C57BL/6J), a strain with only rod function (cpfl1), and a strain with only cone function (rho(-/-)). A stationary flicker light stimulus with intensity ranging from 10(8.6) to 10(15.5) photons/cm2/s was used. We found that the intrinsic signal patterns exhibited stimulus intensity-dependent changes. At a given stimulus intensity, the patterns of intrinsic signals were clearly different in the three strains. These results suggest that the lack of normal functions of certain photoreceptors induces significant reorganization in the visual neural systems in mice.

Outer segment oligomerization of Rds: evidence from mouse models and subcellular fractionation.

Retinal degeneration slow (Rds) is a photoreceptor-specific tetraspanin glycoprotein essential for photoreceptor outer segment (OS) morphogenesis. Over 80 mutations in this protein are associated with several different retinal diseases. Rds forms a mixture of disulfide-linked homomeric dimers, octamers, and higher-order oligomers, with Cys150 playing a crucial role in its oligomerization. Rds also forms noncovalent homo- and hetero-tetramers with its nonglycosylated homologue, Rom-1. Here, we evaluated the subcellular site of Rds oligomerization and the pattern of Rds/Rom-1 complex assembly in several types of knockout mice, including rhodopsin (Rho-/-, lacking rod OS), Rom-1 (Rom-1-/-), neural retina leucine zipper (Nrl-/-, cone-dominant), and in comparison with wild-type (WT, rod-dominant) mice. Oligomerization and the pattern of complex assembly were also evaluated in OS-enriched vs OS-depleted preparations from WT and Rom-1-/- retinas. Velocity sedimentation under reducing- and nonreducing conditions and co-immunoprecipitation experiments showed the presence of Rds mainly as homo- and hetero-tetramers with Rom-1 in the photoreceptor inner segment (IS), while higher-order, disulfide-linked intermediate complexes and oligomers were exclusively present in the photoreceptor OS. Rom-1-independent oligomerization of Rds was observed in Rom-1-/- retinas. The pattern of Rds complexes in cones from Nrl-/- mice was comparable to that in rods from WT mice. On the basis of these findings, we propose that Rds traffics from the IS to the OS as homo- and hetero-tetramers, with subsequent disulfide-linked oligomerization occurring concomitant with OS disc morphogenesis (at either the base of OS or the tip of the connecting cilium). These results suggest that Rds mutations that interfere with tetramer formation can block Rds trafficking to the OS, leading to loss-of-function defects.

Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space.

PURPOSE: To determine whether transplantation of embryonic stem (ES) cells into the subretinal space of rhodopsin-knockout mice has a tumorigenic effect. METHODS: Mouse ES-cell-derived neural precursor cells carrying the sequence for the green fluorescent protein (GFP) gene were grafted subretinally into the eyes of rhodopsin(-/-) mice, whereas control animals underwent sham surgery. Eyes were retrieved after 2, 4, and 8 weeks after cell injection or sham surgery for histologic analysis. RESULTS: Gross morphologic, histologic, and immunohistochemical analysis of eyes at 2 and 4 weeks after engraftment exhibited no morphologic alterations, whereas neoplasia formation was detected in 50% of the eyes evaluated at 8 weeks after engraftment. Because the neoplasias expressed differentiation characteristics of the different germ layers, they were considered to be teratomas. The resultant tumor formation affected almost all layers of the eye, including the retina, the vitreous, and the choroid. CONCLUSIONS: Although ES cells may provide treatment for degenerative disease in the future, their unlimited self-renewal and high differentiation potential poses the risk of tumor induction after engraftment. Thus, more care must be taken before using ES cell transplantation as a therapeutic option for patients with degenerative disease.