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1.  Manipulation of the Recipient Retinal Environment by Ectopic Expression of Neurotrophic Growth Factors Can Improve Transplanted Photoreceptor Integration and Survival 
Cell transplantation  2012;21(5):871-887.
Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP+ve neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.
doi:10.3727/096368911X623871
PMCID: PMC3523316  PMID: 22325046
Photoreceptor; Retina; Transplantation; Neurotrophic factors; Gene therapy; Stem cell
2.  Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors 
Experimental Eye Research  2008;86(4):601-611.
Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.
doi:10.1016/j.exer.2008.01.004
PMCID: PMC2394572  PMID: 18294631
retinal transplantation; Müller cell; outer limiting membrane; cell integration; photoreceptor; stem cells; mouse
3.  Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa 
eLife  2013;2:e00824.
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.
DOI: http://dx.doi.org/10.7554/eLife.00824.001
eLife digest
Retinitis pigmentosa is an inherited disorder in which the gradual degeneration of light-sensitive cells in the outer retina, known as photoreceptors, causes a progressive loss of sight. Retinitis pigmentosa can also occur as part of a wider syndrome: patients with Usher syndrome, for example, suffer from early-onset deafness and then develop retinitis pigmentosa later in life. Usher syndrome is caused by mutations in any of more than ten genes, but the most commonly affected is USH2A, which encodes a protein called usherin. Mutations in USH2A can also cause retinitis pigmentosa on its own.
Clinical trials are underway to determine whether it is possible to treat various forms of inherited retinal degeneration using gene therapy. This involves inserting a functional copy of the gene associated with the disease into an inactivated virus, which is then injected into the eye. The virus carries the target gene to the light-sensitive photoreceptor cells where it can replace the faulty gene. This could be particularly useful for conditions such as Usher syndrome, in which the early-onset deafness makes it possible to diagnose retinitis pigmentosa before substantial numbers of photoreceptor cells have been lost.
For gene therapy to become a widely used strategy for the treatment of retinal degenerative disease, identification and functional interrogation of the disease-causing gene/mutations will be critical. This is especially true for large highly polymorphic genes such as USH2A that often have mutations that are difficult to identify by standard sequencing techniques. Likewise, viruses that can carry large amounts of genetic material, or endogenous genome editing approaches, will need to be developed and validated in an efficient patient-specific model system.
Tucker et al. might have found a way to address these problems. In their study, they used skin cells from a retinitis pigmentosa patient with mutations in USH2A to produce induced pluripotent stem cells. These are cells that can be made to develop into a wide variety of mature cell types, depending on the exact conditions in which they are cultured. Tucker et al. used these stem cells to generate photoreceptor precursor cells, which they transplanted into the retinas of immune-suppressed mice. The cells developed into normal-looking photoreceptor cells that expressed photoreceptor-specific proteins.
These results have several implications. First, they support the idea that stem cell-derived retinal photoreceptor cells, generated from patients with unknown mutations, can be used to identify disease-causing genes and to interrogate disease pathophysiology. This will allow for a more rapid development of gene therapy strategies. Second, they demonstrate that USH2A mutations cause retinitis pigmentosa by affecting photoreceptors later in life rather than by altering their development. This suggests that it should, via early intervention, be possible to treat retinitis pigmentosa in adult patients with this form of the disease. Third, the technique could be used to generate animal models in which to study the effects of specific disease-causing mutations on cellular development and function. Finally, this study suggests that skin cells from adults with retinitis pigmentosa could be used to generate immunologically matched photoreceptor cells that can be transplanted back into the same patients to restore their sight. Many questions remain to be answered before this technique can be moved into clinical trials but, in the meantime, it will provide a new tool for research into this major cause of blindness.
DOI: http://dx.doi.org/10.7554/eLife.00824.002
doi:10.7554/eLife.00824
PMCID: PMC3755341  PMID: 23991284
next-generation sequencing; retinal degeneration; induced pluripotent stem cells; retinal transplantation; retinal cell differentiation; retinitis pigmentosa; Human; Mouse
4.  Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells 
Cell Research  2013;23(6):788-802.
Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin+Sox2+Pax6+ multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.
doi:10.1038/cr.2013.48
PMCID: PMC3674387  PMID: 23567557
retinal stem cells; photoreceptor cells
5.  The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells? 
Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS) cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE), to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using gene-directed reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.
PMCID: PMC4074479  PMID: 24982737
Photoreceptor; Regeneration; Replacement; Retina; Retinal Pigment Epithelium
6.  Long-term survival and differentiation of retinal neurons derived from human embryonic stem cell lines in un-immunosuppressed mouse retina 
Molecular Vision  2012;18:920-936.
Purpose
To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression.
Methods
hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration 1 and wingless) blocking morphogens Dickkopf-1 (Dkk-1) and Insulin-like growth factor 1 (IGF-1) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4–6-week-old) mice (C57BL/6J x129/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina.
Results
At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2 (LHX2), early NR markers (Ceh-10 homeodomain containing homolog [CHX10], achaete-scute complex homolog 1 [MASH1], mouse atonal homolog 5 [MATH5], neurogenic differentiation 1 [NEUROD1]), and some retinal cell fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], prospero homeobox 1 [PROX1], and recoverin). The cells in the subretinal grafts matured to predominantly recoverin [+] phenotype by 3 months and survived in a xenogenic environment without immunosuppression as long as the blood–retinal barrier was not breached by the transplantation procedure. The epiretinal grafts survived but did not express markers of mature retinal cells. Retinal integration into the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) was efficient from the epiretinal but not subretinal grafts. The subretinal grafts showed limited ability to structurally integrate into the host retina and only in cases when NR was damaged during grafting. Only limited synaptogenesis and no tumorigenicity was observed in grafts.
Conclusions
Our studies show that (i) immunosuppression is not mandatory to xenogenic graft survival in the retina, (ii) the subretinal but not the epiretinal niche can promote maturation of hESC-RPCs to photoreceptors, and (iii) the hESC-RPCs from epiretinal but not subretinal grafts can efficiently integrate into the RGC layer and INL. The latter could be of value for long-lasting neuroprotection of retina in some degenerative conditions and glaucoma. Overall, our results provide new insights into the technical aspects associated with cell-based therapy in the retina.
PMCID: PMC3335781  PMID: 22539871
7.  Generation of Functional Eyes from Pluripotent Cells 
PLoS Biology  2009;7(8):e1000174.
The directed differentiation of pluripotent cells into specific cell-types is a major hurdle in regenerative medicine. This study shows the eye field transcription factor factors can direct pluripotent cells into functioning frog eyes.
Pluripotent cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells are the starting point from which to generate organ specific cell types. For example, converting pluripotent cells to retinal cells could provide an opportunity to treat retinal injuries and degenerations. In this study, we used an in vivo strategy to determine if functional retinas could be generated from a defined population of pluripotent Xenopus laevis cells. Animal pole cells isolated from blastula stage embryos are pluripotent. Untreated, these cells formed only epidermis, when transplanted to either the flank or eye field. In contrast, misexpression of seven transcription factors induced the formation of retinal cell types. Induced retinal cells were committed to a retinal lineage as they formed eyes when transplanted to the flanks of developing embryos. When the endogenous eye field was replaced with induced retinal cells, they formed eyes that were molecularly, anatomically, and electrophysiologically similar to normal eyes. Importantly, induced eyes could guide a vision-based behavior. These results suggest the fate of pluripotent cells may be purposely altered to generate multipotent retinal progenitor cells, which differentiate into functional retinal cell classes and form a neural circuitry sufficient for vision.
Author Summary
The goal of regenerative medicine is to replace dead or dying cells. Successful cell replacement depends on the ability of donor cells to differentiate into all functional cell types lost in the target organ. Blindness resulting from retinal disease or damage, for example, would require the replacement of as many as seven specialized cell types found in the retina. The most celebrated characteristic of pluripotent cells is their ability to differentiate into any adult cell type. This defining feature, however, presents the challenge of identifying the conditions for their conversion to the cell types needed for tissue repair. We asked if pluripotent cells could be directed to generate all the retinal cell types necessary to form a functional eye in the frog, Xenopus laevis. If left untreated, transplanted pluripotent cells only form the epidermal layer of the skin. However, when forced to express the eye field transcription factor (EFTF) genes, the cells differentiate into all seven retinal cell classes and eventually organize themselves into a functioning eye that can detect light and guide tadpoles in a vision-based behavior. Our results demonstrate that pluripotent cells can be purposely altered to generate all the functional retinal cell classes necessary for sight.
doi:10.1371/journal.pbio.1000174
PMCID: PMC2716519  PMID: 19688031
8.  Transplantation of Photoreceptor and Total Neural Retina Preserves Cone Function in P23H Rhodopsin Transgenic Rat 
PLoS ONE  2010;5(10):e13469.
Background
Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.
Methods and Findings
We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.
Conclusions
We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.
doi:10.1371/journal.pone.0013469
PMCID: PMC2957406  PMID: 20976047
9.  Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina 
Nature biotechnology  2013;31(8):10.1038/nbt.2643.
Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair1 and restoration of vision through transplantation of photoreceptor precursors obtained from post-natal retinas into visually impaired adult mice2,3. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages4-6. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs6. We show that Rhop.GFP-selected rod precursors derived by this protocol integrate within degenerate retinae of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently than cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
doi:10.1038/nbt.2643
PMCID: PMC3826328  PMID: 23873086
10.  Gene expression changes during retinal development and rod specification 
Molecular Vision  2015;21:61-87.
Purpose
Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3–5 (P3–5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors.
Methods
Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR).
Results
Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes’ promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random.
Conclusions
This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).
PMCID: PMC4300221
11.  Gene expression changes during retinal development and rod specification 
Molecular Vision  2015;21:61-87.
Purpose
Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3–5 (P3–5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors.
Methods
Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR).
Results
Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes’ promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random.
Conclusions
This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).
PMCID: PMC4301594
12.  Survival and Migration of Pre-induced Adult Human Peripheral Blood Mononuclear Cells in Retinal Degeneration Slow (rds) Mice Three Months After Subretinal Transplantation 
Introduction: Retinitis pigmentosa (RP), an inherited disease characterized by progressive loss of photoreceptors and retinal pigment epithelium, is a leading genetic cause of blindness. Cell transplantation to replace lost photoreceptors is a potential therapeutic strategy, but technical limitations have prevented clinical application. Adult human peripheral blood mononuclear cells (hPBMCs) may be an ideal cell source for such therapies. This study examined the survival and migration of pre-induced hPBMCs three months after subretinal transplantation in the retinal degeneration slow (rds) mouse model of RP. Materials and Methods: Freshly isolated adult hPBMCs were pre-induced by co-culture with neonatal Sprague-Dawley (SD) rat retinal tissue for 4 days in neural stem cell medium. Pre-induced cells were labeled with CM-DiI for tracing and injected into the right subretinal space of rds mice by the trans-scleral approach. After two and three months, right eyes were harvested and transplanted cell survival and migration examined in frozen sections and whole mountretinas. Immunofluorescence in whole-mount retinas was used to detect the expression of human neuronal and photorece ptorsprotein markers by transplanted cells. Results: Pre-induced adult hPBMCs could survive in vivo and migrate to various parts of the retina. After two and three months, transplanted cells were observed in the ciliary body, retinal outer nuclear layer, inner nuclear layer, ganglion cell layer, optic papilla, and within the optic nerve. The neuronal and photoreceptor markers CD90/Thy1, MAP-2, nestin, and rhodopsin were expressed by subpopulations of CM-DiI-positive cells three months after subretinal transplantation. Conclusion: Pre-induced adult hPBMCs survived for at least three months after subretinal transplantation, migrated throughout the retina, and expressed human protein markers. These results suggest that hPBMCs could be used for cell replacement therapy to treat retinal degenerative diseases.
doi:10.2174/1574888X09666131219115125
PMCID: PMC4101734  PMID: 24350910
Adult stem cells; hPBMCs; migration; retinal degeneration; subretinal transplantation; survival.
13.  Cell replacement and visual restoration by retinal sheet transplants 
Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a ‘nursing’ role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance – they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity.
In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy.
doi:10.1016/j.preteyeres.2012.06.003
PMCID: PMC3472113  PMID: 22771454
retinal degeneration; retinal transplantation; retinal progenitor sheets; electrophysiology; superior colliculus; trans-synaptic tracing; electron microscopy
14.  Defining the integration capacity of ES cell-derived photoreceptor precursors 
Stem cells (Dayton, Ohio)  2012;30(7):1424-1435.
Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human ES and iPS cells, potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ES cells with defined factors. In this study we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ES cell-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx+ and Rhodopsin+ photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ES cell-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ES cell-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ES cell-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ES cell-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.
doi:10.1002/stem.1123
PMCID: PMC3580313  PMID: 22570183
Embryonic stem cells; Retina; Cell transplantation; Photoreceptor cells; Fluorescent protein reporter genes; Stem cell transplantation
15.  Transplantation of human embryonic stem cells derived photoreceptors restores some visual function in Crx deficient mice 
Cell stem cell  2009;4(1):73-79.
Summary
Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina; photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem (ES) cells could in principle provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ES cells. We now show that retinal cells derived from human ES cells will migrate into mouse retinas following intra-ocular injection, settle into the appropriate layers and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx -/- mice (a model of Leber's Congenital Amaurosis), the hES cell derived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hES cells can, in principle, be used for photoreceptor replacement therapies.
doi:10.1016/j.stem.2008.10.015
PMCID: PMC2713676  PMID: 19128794
16.  Genetically Modified Neural Stem Cells for a Local and Sustained Delivery of Neuroprotective Factors to the Dystrophic Mouse Retina 
Stem Cells Translational Medicine  2013;2(12):1001-1010.
In two mouse models of retinitis pigmentosa, intravitreal transplantations of adherently cultivated neural stem cells that had been modified to secrete ciliary neurotrophic factor resulted in a sustained delivery of functionally relevant quantities of the cytokine to the dystrophic retina. These transplantations may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based administration of neurotrophic factors in mouse models of photoreceptor degeneration.
A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6brd1 and Pde6brd10 mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6brd1 mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration.
doi:10.5966/sctm.2013-0013
PMCID: PMC3841080  PMID: 24167317
Neural stem cell; Stem cell transplantation; Retinal photoreceptors; Lentiviral vector; Ciliary neurotrophic factor
17.  Effective Transplantation of Photoreceptor Precursor Cells Selected Via Cell Surface Antigen Expression 
Stem cells (Dayton, Ohio)  2011;29(9):1391-1404.
Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome-conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP-expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence-activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild-type or Crb1rd8/rd8 or Prph2rd2/rd2 mouse eye. The CD73/CD24-sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18-fold higher integration efficiency than that of unsorted cells and 2.3-fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof-of-principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donor photoreceptor precursor cells can increase the number of integrated photoreceptor cells, which is a prerequisite for the restoration of sight.
doi:10.1002/stem.694
PMCID: PMC3303132  PMID: 21774040
Retina; Cell transplantation; Cell surface markers; fluorescence-activated cell sorting; Stem cell transplantation; Fluorescent protein reporter genes; Microarray; Embryonic stem cells
18.  Embryonic Stem Cell-Derived Neural Progenitors Incorporate into Degenerating Retina and Enhance Survival of Host Photoreceptors 
Stem Cells (Dayton, Ohio)  2005;24(2):274-283.
Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type–specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.
doi:10.1634/stemcells.2005-0059
PMCID: PMC3381839  PMID: 16123383
Embryonic stem cell; Retina; Transplantation; mnd mouse; Differentiation; Repair; Neuronal ceroid lipofuscinoses
19.  Long-Term Survival of Photoreceptors Transplanted into the Adult Murine Neural Retina Requires Immune Modulation 
Stem cells (Dayton, Ohio)  2010;28(11):1997-2007.
Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp+ve photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.
doi:10.1002/stem.520
PMCID: PMC3272388  PMID: 20857496
Stem cell; Progenitor cell; Photoreceptor; Retina; Cell transplantation; Immune response
20.  In Vitro Expanded Stem Cells from the Developing Retina Fail to Generate Photoreceptors but Differentiate into Myelinating Oligodendrocytes 
PLoS ONE  2012;7(7):e41798.
Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive ‘retinal stem cells’ (‘RSCs’) can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, ‘RSCs’, by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, ‘RSCs’ can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that ‘RSCs’ expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina.
doi:10.1371/journal.pone.0041798
PMCID: PMC3405018  PMID: 22848612
21.  Protection of Visual Functions by Human Neural Progenitors in a Rat Model of Retinal Disease 
PLoS ONE  2007;2(3):e338.
Background
A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat.
Methodology/Principal Findings
Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90–100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed.
Conclusions/Significance
Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo.
doi:10.1371/journal.pone.0000338
PMCID: PMC1828619  PMID: 17396165
22.  Intraretinal Transplantation for Rod-Cell Replacement in Light-Damaged Retinas 
Blindness from retinal disease is often the consequence of extensive damage to the photoreceptor cell population, while other cell types which form the neural retina are relatively spared. In this setting, transplantation of photoreceptor cells could offer hope for the restoration of some degree of visual function. We testd the feasibility of this approach by transplanting immature retinal cells into the eyes of adult rats affected by late stage phototoxic retinopathy, which are almost totally devoid of photoreceptor cells.
Dissociated neuroretinal cells from newborn rats were injected into the hosts' retinas. These cells were labelled with the fluorescent tracer Fast-blue for identification within the host eye. Survival time ranged from 3 to 100 post-transplantation days.
Fundus examination of light-irradiated eyes showed pallor caused by a considerable reduction of the retino-choroidal vascular bed after light irradiation. Histologically the hosts exhibited decimation of the elements forming the outer layers.throughout the entire retina.
As visualized by light and electron microscopic procedures, we report the differentiation of clusters of transplanted photoreceptor cells, and the integration of these cells within the adjacent areas of the host retina.
Fluorescence microscopy showed these clusters to be formed by fluorescently labelled cells developing in intimate contact with the unlabelled host retina. Electron microscopically it was possible to determine that these photoreceptors had established synaptic contacts. These observations indicate that successful transplantation of immature retinal cells is feasible into adult eyes that have suffered extensive retino-choroidal damage. These findings also support the concept that retinal transplantation is a procedure which may open new avenues into the study of retinal repair.
doi:10.1155/NP.1989.1
PMCID: PMC2564997  PMID: 2519517
23.  Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells 
Human Molecular Genetics  2010;19(23):4545-4559.
Retinal degenerative disease causing loss of photoreceptor cells is the leading cause of untreatable blindness in the developed world, with inherited degeneration affecting 1 in 3000 people. Visual acuity deteriorates rapidly once the cone photoreceptors die, as these cells provide daylight and colour vision. Here, in proof-of-principle experiments, we demonstrate the feasibility of cone photoreceptor transplantation into the wild-type and degenerating retina of two genetic models of Leber congenital amaurosis, the Crb1rd8/rd8 and Gucy2e−/− mouse. Crx-expressing cells were flow-sorted from the developing retina of CrxGFP transgenic mice and transplanted into adult recipient retinae; CrxGFP is a marker of cone and rod photoreceptor commitment. Only the embryonic-stage Crx-positive donor cells integrated within the outer nuclear layer of the recipient and differentiated into new cones, whereas postnatal cells generated a 10-fold higher number of rods compared with embryonic-stage donors. New cone photoreceptors displayed unambiguous morphological cone features and expressed mature cone markers. Importantly, we found that the adult environment influences the number of integrating cones and favours rod integration. New cones and rods were observed in ratios similar to that of the host retina (1:35) even when the transplanted population consisted primarily of cone precursors. Cone integration efficiency was highest in the cone-deficient Gucy2e−/− retina suggesting that cone depletion creates a more optimal environment for cone transplantation. This is the first comprehensive study demonstrating the feasibility of cone transplantation into the adult retina. We conclude that flow-sorted embryonic-stage Crx-positive donor cells have the potential to replace lost cones, as well as rods, an important requirement for retinal disease therapy.
doi:10.1093/hmg/ddq378
PMCID: PMC2972691  PMID: 20858907
24.  Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina 
Stem cells (Dayton, Ohio)  2011;29(6):972-980.
Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments utilizing stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with swine induced pluripotent stem cells (iPSC). Here, we subjected swine iPSC to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of RHO and ROM1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that swine iPSC can differentiate into photoreceptors in culture and these cells can integrate into the damaged swine neural retina thus laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.
doi:10.1002/stem.637
PMCID: PMC4263955  PMID: 21491544
photoreceptor differentiation; swine retina; induced pluripotent stem cells; retinal transplantation
25.  Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig 
Stem Cells International  2012;2012:939801.
Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed.
doi:10.1155/2012/939801
PMCID: PMC3337587  PMID: 22567027

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