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Genotyping assays of immunodeficient S334ter-3 rats. (a) S334ter transgene genotyping assay. Lane 1 15 bp–3 kb size marker. Lane 2 transgene-negative sample. Lane 3 transgene-positive sample. Sizes in base pairs (bp) are indicated to the left of the image. An amplicon of 350 bp indicates the presence of the transgene. The 15- and 3000-bp alignment markers are present in all lanes. (b) Allelic discrimination assay plot for detection of the Foxn1rnu mutation. The fluorescence levels of VIC (wild type, allele X) and FAM (mutant, allele Y) are plotted on the x-axis and y-axis, respectively. The genotypes of each sample are represented by blue diamonds (homozygous Foxn1rnu), red circles (homozygous for the wild-type Foxn1 allele), or green triangles (heterozygous +/Foxn1rnu). The no template negative control is represented by the gray box. Reprinted from Graefes Arch Clin Exp Ophthalmol, vol. 252, Seiler et al., a new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression, pages 1079–1092, copyright (2014), with permission from Elsevier. DOI 10.1007/s00417-014-2638-y.
Source publication
Dysfunction and death of retinal pigment epithelium (RPE) and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula). Further, s...
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Citations
... This may lead to constant variations in the viewing distance (distance between the eye and the visual stimuli). Since the rat's vision is sensitive to variation in the viewing distance 22 , its free movement inside the testing chamber may cause variations in the visual acuity scores. High variability in the visual acuity score within the same group of animals or in the same animal between different tests can cause difficulties in obtaining meaningful statistical inference. ...
Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits movement so that the rat's head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat's visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.
... According to several studies, the morphology and function of iPSC-derived RPE cells were similar to naïve RPE in vivo and in vitro [71]. Despite progress in stem cell research, scientists are faced with different challenges such as ethical issues, regulatory controversies, safety, and efficacy, along with the technical difficulties of adjusting this method into a standard approach for clinical application. ...
... Despite progress in stem cell research, scientists are faced with different challenges such as ethical issues, regulatory controversies, safety, and efficacy, along with the technical difficulties of adjusting this method into a standard approach for clinical application. Before iPSCs can be considered a reliable cell source for clinical-grade purposes, a variety of concerns should be taken into consideration [71]. For instance, transplanting autologous adipose stem cells, with minimal evidence of safety or efficacy, into the eye of three patients with severe AMD caused vitreous hemorrhage, PVR formation, ocular hypertension, retinal detachment through neovascularization, and lens displacement [71]. ...
... Before iPSCs can be considered a reliable cell source for clinical-grade purposes, a variety of concerns should be taken into consideration [71]. For instance, transplanting autologous adipose stem cells, with minimal evidence of safety or efficacy, into the eye of three patients with severe AMD caused vitreous hemorrhage, PVR formation, ocular hypertension, retinal detachment through neovascularization, and lens displacement [71]. iPSC-RPE transplantation is the most challenging procedure due to immune responses. ...
Retinal pigment epithelium (RPE) degeneration is the hallmark of age-related macular degeneration (AMD). AMD, as one of the most common causes of irreversible visual impairment worldwide, remains in need of an appropriate approach to restore retinal function. Wet AMD, which is characterized by neovascular formation, can be stabilized by currently available therapies, including laser photocoagulation, photodynamic therapy, and intraocular injections of anti-VEFG (anti-vascular endothelial growth factor) therapy or a combination of these modalities. Unlike wet AMD, there is no effective therapy for progressive dry (non-neovascular) AMD. However, stem cell-based therapies, a part of regenerative medicine, have shown promising results for retinal degenerative diseases such as AMD. The goal of RPE cell therapy is to return the normal structure and function of the retina by re-establishing its interaction with photoreceptors, which is essential to vision. Considering the limited source of naturally occurring RPE cells, recent progress in stem cell research has allowed the generation of RPE cells from human pluripotent cells, both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC). Since iPSCs face neither ethical arguments nor significant immunological considerations when compared to ESCs, they open a new horizon for cell therapy of AMD. The current study aims to discuss AMD, review the protocols for making human iPSCs-derived RPEs, and summarize recent developments in the field of iPSC-derived RPEs cell therapy.
... In this regard, we first assessed the cell integrity and purity of our hiPSC-RPE cultures to exclude undifferentiated cells using a protocol of cell sorting and purification by extensive culture and passaging. Upon transplantation, there was no evidence of neoplastic or teratoma structures or any other adverse event in any of the transplanted porcine eyes studied, in accordance with other studies in different animal models and in humans [12,58,59,64]. Furthermore, there was no evidence of endophthalmitis, retinal edema, uveitis, epiretinal membrane, proliferative vitreoretinopathy, encapsulation, or retinal rupture in the immunosuppressed animals. ...
Background:
The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA).
Methods:
Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology.
Results:
In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer.
Conclusions:
These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.
... Other large mammals such as cats [45] and pigs [46,47] have been used for pre-clinical evaluation of retinal sheets, RPE monolayer and photoreceptor precursors but are also not ideal. Limitations of the cat model include the presence of the reflective tapetum lucidum, [48], while porcine eyes lack a fovea and are not optimal for pre-clinical studies due to inconsistencies in disease development patterns [48,49]. In contrast, the ocular anatomy of NHPs resemble humans the most, making them most suitable for the evaluation of retinal cell-based therapies [44,[50][51][52][53]. ...
... Other large mammals such as cats [45] and pigs [46,47] have been used for pre-clinical evaluation of retinal sheets, RPE monolayer and photoreceptor precursors but are also not ideal. Limitations of the cat model include the presence of the reflective tapetum lucidum, [48], while porcine eyes lack a fovea and are not optimal for pre-clinical studies due to inconsistencies in disease development patterns [48,49]. In contrast, the ocular anatomy of NHPs resemble humans the most, making them most suitable for the evaluation of retinal cell-based therapies [44,[50][51][52][53]. ...
Background
Retinal regenerative therapies hold great promise for the treatment of inherited retinal degenerations (IRDs). Studies in preclinical lower mammal models of IRDs have suggested visual improvement following retinal photoreceptor precursors transplantation, but there is limited evidence on the ability of these transplants to rescue retinal damage in higher mammals. The purpose of this study was to evaluate the therapeutic potential of photoreceptor precursors derived from clinically compliant induced pluripotent stem cells (iPSCs).
Methods
Photoreceptor precursors were sub-retinally transplanted into non-human primates ( Macaca fascicularis ). The cells were transplanted both in naïve and cobalt chloride-induced retinal degeneration models who had been receiving systemic immunosuppression for one week prior to the procedure. Optical coherence tomography, fundus autofluorescence imaging, electroretinography, ex vivo histology and immunofluorescence staining were used to evaluate retinal structure, function and survival of transplanted cells.
Results
There were no adverse effects of iPSC-derived photoreceptor precursors on retinal structure or function in naïve NHP models, indicating good biocompatibility. In addition, photoreceptor precursors injected into cobalt chloride-induced retinal degeneration NHP models demonstrated an ability both to survive and to mature into cone photoreceptors at 3 months post-transplant. Optical coherence tomography showed restoration of retinal ellipsoid zone post-transplantation.
Conclusions
These findings demonstrate the safety and therapeutic potential of clinically compliant iPSC-derived photoreceptor precursors as a cell replacement source for future clinical trials.
... In the first case, to avoid molecules/cells dispersion into the host tissue, encapsulation can be employed. Porous materials permits the signals of the damaged environment to reach the implanted cells and stimulate the production of neuroprotective biomolecules, as well as to allow the diffusion of the secreted molecules to the damaged tissue (Battler and Leor, 2006;Weiner, 2008;Seiler and Aramant, 2012;Lin et al., 2017;Fernández-García et al., 2018;González-Nieto et al., 2018). However, although neuroprotective factors could protect retinal cells before apoptosis occurrence, their application is not a viable solution for many patients with advanced retinal pathologies. ...
Age-related Macular Degeneration (AMD) is an up-to-date untreatable chronic neurodegenerative eye disease of multifactorial origin, and the main causes of blindness in over 65 y.o. people. It is characterized by a slow progression and the presence of a multitude of factors, highlighting those related to diet, genetic heritage and environmental conditions, present throughout each of the stages of the illness. Current therapeutic approaches, mainly consisting on intraocular drug delivery, are only used for symptoms relief and/or to decelerate the progression of the disease. Furthermore, they are overly simplistic and ignore the complexity of the disease and the enormous differences in the symptomatology between patients. Due to the wide impact of the AMD and the up-to-date absence of clinical solutions, Due to the wide impact of the AMD and the up-to-date absence of clinical solutions, different treatment options have to be considered. Cell therapy is a very promising alternative to drug-based approaches for AMD treatment. Cells delivered to the affected tissue as a suspension have shown poor retention and low survival rate. A solution to these inconveniences has been the encapsulation of these cells on biomaterials, which contrive to their protection, gives them support, and favor their retention of the desired area. We offer a two-papers critical review of the available and under development AMD therapeutic approaches, from a biomaterials and biotechnological point of view. We highlight benefits and limitations and we forecast forthcoming alternatives based on novel biomaterials and biotechnology methods. In this second part we review the preclinical and clinical cell-replacement approaches aiming at the development of efficient AMD-therapies, the employed cell types, as well as the cell-encapsulation and cell-implant systems. We discuss their advantages and disadvantages and how they could improve the survival and integration of the implanted cells.
... To overcome the lethal side effect of systemic delivery of retinotoxic reagents or random distribution of the retinal lesion after their intravitreal delivery [15,22], and to facilitate the study of potential therapeutic interventions, subretinal stem cell transplantation in particular, we subretinally injected 100 µL of SNP solutions at three doses (0.05, 0.1, and 0.2 mM) or NS into the posterior pole of the monkey retina next to the fovea under a surgical microscope ( Figure 1A). Within 1 h after subretinal injection (SI), SD-OCT images showed that SNP solutions or NS caused a hypo-reflective subretinal bleb, indicating the successful delivery ( Figure 1B). ...
... Several approaches, such as intravenous injection, intravitreal injection, and SI, have been applied to deliver retinotoxic reagents to animal eyes to develop RD-like models [15,17,19,22,28,[32][33][34][35][36][37]. Each approach has its own advantages and disadvantages. ...
... Intravenous injection is a common and easy method of damaging the bilateral retina with large lesion areas in small-and medium-sized animals [17,25,32,33]. However, systemic administration of retinotoxic reagents also affects the general health status of the experimental animals and can even lead to death and tumor formation [15,22,36,37]. ...
Background: Retinal degenerative disorders (RDs) are the main cause of blindness without curable treatment. Our previous studies have demonstrated that human induced pluripotent stem cells can differentiate into retinal organoids with all subtypes of retina, which provides huge promises for treating these diseases. Before it can be turned into reality, RD animal models are required to evaluate the safety and efficacy of stem cell therapy, and to develop the surgical tools and procedures for cell transplantation in patients. This study is to develop a monkey model of RD with controllable of lesion sites which can be rapidly prepared, for the studies of preclinical stem cell therapy among other applications.
Methods: Sodium nitroprusside (SNP) in three doses was delivered into the monkey eye by subretinal injection (SI) and normal saline was applied as control. Structural and functional changes of the retinas were evaluated via multimodal imaging techniques and multifocal electroretinography (mfERG) before and after the treatment. Histological examination was performed to identify the target layer of the affected retina. The health status of monkeys was monitored during the experiment.
Results: Well defined lesion with various degree of retinal degeneration was established at the posterior pole of retina as early as 7 days after SNP SI. The damage effect of SNP was dose-dependent. 0.05 mM SNP caused invisible structural changes in retina, similar to the control. 0.1 mM SNP led to the loss of outer retinal layer, including OPL, ONL and RPE, while 0.2 mM SNP impacted the entire layer of retina and choroid. MfERG showed reduced amplitude in the damaged region. The structural and functional damages were not recovered after 7-month follow-up.
Conclusion: A simple, rapidly induced, lesion site-controllable, retinal degeneration model in monkey was established by the subretinal injection of 0.1 mM SNP. This monkey model closely mimics the histological changes of RDs, and provides a valuable platform for preclinical assessment of stem cell therapy.
... To overcome the lethal side effect of systemic delivery of retinotoxic reagents or random distribution of the retinal lesion after their intravitreal delivery [15,22], and to facilitate the study of potential therapeutic interventions, subretinal stem cell transplantation in particular, we subretinally injected 100 µL of SNP solutions at three doses (0.05, 0.1, and 0.2 mM) or NS into the posterior pole of the monkey retina next to the fovea under a surgical microscope ( Figure 1A). Within 1 h after subretinal injection (SI), SD-OCT images showed that SNP solutions or NS caused a hypo-reflective subretinal bleb, indicating the successful delivery ( Figure 1B). ...
... Several approaches, such as intravenous injection, intravitreal injection, and SI, have been applied to deliver retinotoxic reagents to animal eyes to develop RD-like models [15,17,19,22,28,[32][33][34][35][36][37]. Each approach has its own advantages and disadvantages. ...
... Intravenous injection is a common and easy method of damaging the bilateral retina with large lesion areas in small-and medium-sized animals [17,25,32,33]. However, systemic administration of retinotoxic reagents also affects the general health status of the experimental animals and can even lead to death and tumor formation [15,22,36,37]. ...
Background:
Retinal degenerative disorders (RDs) are the main cause of blindness without curable treatment. Our previous studies have demonstrated that human-induced pluripotent stem cells can differentiate into retinal organoids with all subtypes of retina, which provides huge promise for treating these diseases. Before these methods can be realized, RD animal models are required to evaluate the safety and efficacy of stem cell therapy and to develop the surgical tools and procedures for cell transplantation in patients. This study involved the development of a monkey model of RD with controllable lesion sites, which can be rapidly prepared for the study of preclinical stem cell therapy among other applications.
Methods:
Sodium nitroprusside (SNP) in three doses was delivered into the monkey eye by subretinal injection (SI), and normal saline was applied as control. Structural and functional changes of the retinas were evaluated via multimodal imaging techniques and multifocal electroretinography (mfERG) before and after the treatment. Histological examination was performed to identify the target layer of the affected retina. The health status of monkeys was monitored during the experiment.
Results:
Well-defined lesions with various degrees of retinal degeneration were induced at the posterior pole of retina as early as 7 days after SNP SI. The damage of SNP was dose dependent. In general, 0.05 mM SNP caused mild structural changes in the retina; 0.1 mM SNP led to the loss of outer retinal layers, including the outer plexiform layer (OPL), outer nuclear layer (ONL), and retinal pigment epithelium (RPE); while 0.2 mM SNP impacted the entire layer of the retina and choroid. MfERG showed reduced amplitude in the damaged region. The structural and functional damages were not recovered at 7-month follow-up.
Conclusion:
A rapidly induced lesion site-controllable retinal degeneration monkey model was established by the subretinal administration of SNP, of which the optimal dose is 0.1 mM. This monkey model mimics the histological changes of advanced RDs and provides a valuable platform for preclinical assessment of stem cell therapy for RDs.
... E ye is a meticulous organ with special immune environment which is established through the blood-aqueous barrier and the blood-retina barrier [1][2] . The immune system protects eyes from dangerous immune reactions, for example, it can wipe out activated cells that might invade the eye and destroy vision by doing damage to important structures such as the retina and local ocular defense system including vitreous cavity [3][4] . Heat shock proteins (HSP) belong to a family of proteins which are conservative in evolution and inducible by variety of stress in expression. ...
Aim:
To investigate the relationships between the changes of heat shock protein 27 antibody (anti-HSP27) in serum/cerebrospinal fluid (CSF), intraocular pressure (IOP), retinal ganglion cell (RGC) apoptosis in a rat glaucoma model and disclose the underlying pathogenesis of glaucoma.
Methods:
A total of 115 Wistar rats were randomly divided into 4 groups. Group 1 was the ocular hypertension group by condensing 3 episcleral & limbal veins or episcleral area of right eye (HP group, n=25) and sham operation group with conjunctiva incision without coagulation (n=25). Group 2: HSP27 or dose-matched PBS was injected into the vitreous (V-HSP27 group, n=15; V-PBS group, n=15). Group 3: HSP27 and complete Freund's adjuvant or dose-matched PBS was injected subcutaneously into the hind limb accompanied intraperitoneal injection of pertussis toxin [sensitized group (I-HSP27 group), n=15; I-PBS group, n=15)]. Group 4 was normal group without any treatment (n=5). IOPs of the rats were measured before, day 3, weeks 1, 2, 4, 6, and 8 after treatment. Paraffin-embedded sections were prepared for HE staining and RGCs apoptosis were detected by TUNEL. Anti-HSP27 level in serum and CSF were examined by ELISA.
Results:
IOPs were elevated significantly in HP and V-HSP27, V-PBS groups (P<0.01) and positively related to anti-HSP27 levels in serum and CSFs. Anti-HSP27 levels in serum and CSF were elevated significantly in I-HSP27 group compared to other groups (P<0.05). However, the IOPs did not show any relationship with the high-level anti-HSP27 in serum and CSFs. RGC apoptosis were all elevated significantly in the HP, V-HSP27, V-PBS and I-HSP27 groups and also positively relative with anti-HSP27 level in serum and CSFs except that high-level of anti-HSP27 in the serum of I-HSP group.
Conclusion:
The increases of anti-HSP27 levels in serum and CSFs both promote IOP escalation and the increase of RGC apoptosis in retina when anti-HSP27 is at low level. The case of high-level anti-HSP27 is opposite and shows protective function in preventing IOP increase and RGC apoptosis.
... As AMD progresses, these interfaces also become damaged, disrupting the retinal homoeostasis and leading to severe vision loss. In such cases, recovery of the lost vision may only be achieved by retinal transplantation, but the poor availability of allografts, together with the high risk of immune rejection, make this a rather limited solution [5] . ...
In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration.
In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact.
Statement of significance
Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.
... The dystrophic Royal College of Surgeons (RCS) rats [10,11] and various lines of transgenic S334ter rats [12][13][14] are the most commonly used rat disease models in ophthalmic research. These models are commonly used for the assessment of beneficial effects of cell replacement therapies [15][16][17][18][19][20][21][22]. The dystrophic RCS rats are characterized by RPE dysfunction due to the deletion in the Mer tyrosine kinase (MerTK) receptor that abolishes internalization of PR outer segments by RPE cells [23]. ...
Purpose
To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats.
Methods
Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control.
Results
Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1–3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection.
Conclusions
This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.
