RESUMEN
Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.
Asunto(s)
Células Madre Pluripotentes Inducidas , Macaca fascicularis , Epitelio Pigmentado de la Retina , Animales , Epitelio Pigmentado de la Retina/trasplante , Epitelio Pigmentado de la Retina/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Degeneración Macular/patologíaAsunto(s)
Hemorragia Retiniana , Epitelio Pigmentado de la Retina , Tomografía de Coherencia Óptica , Agudeza Visual , Degeneración Macular Húmeda , Humanos , Degeneración Macular Húmeda/diagnóstico , Hemorragia Retiniana/diagnóstico , Hemorragia Retiniana/etiología , Hemorragia Retiniana/cirugía , Enfermedad Aguda , Epitelio Pigmentado de la Retina/trasplante , Epitelio Pigmentado de la Retina/patología , Masculino , Estudios de Seguimiento , Femenino , Angiografía con Fluoresceína/métodos , Anciano , Células Madre Embrionarias Humanas/trasplante , Factores de Tiempo , Gravedad del Paciente , Trasplante de Células Madre/métodosRESUMEN
PURPOSE: To report long-term results from a phase 1/2a clinical trial assessment of a scaffold-based human embryonic stem cell-derived retinal pigmented epithelium (RPE) implant in patients with advanced geographic atrophy (GA). DESIGN: A single-arm, open-label phase 1/2a clinical trial approved by the United States Food and Drug Administration. PARTICIPANTS: Patients were 69-85 years of age at the time of enrollment and were legally blind in the treated eye (best-corrected visual acuity [BCVA], ≤ 20/200) as a result of GA involving the fovea. METHODS: The clinical trial enrolled 16 patients, 15 of whom underwent implantation successfully. The implant was administered to the worse-seeing eye with the use of a custom subretinal insertion device. The companion nonimplanted eye served as the control. The primary endpoint was at 1 year; thereafter, patients were followed up at least yearly. MAIN OUTCOME MEASURES: Safety was the primary endpoint of the study. The occurrence and frequency of adverse events (AEs) were determined by scheduled eye examinations, including measurement of BCVA and intraocular pressure and multimodal imaging. Serum antibody titers were collected to monitor systemic humoral immune responses to the implanted cells. RESULTS: At a median follow-up of 3 years, fundus photography revealed no migration of the implant. No unanticipated, severe, implant-related AEs occurred, and the most common anticipated severe AE (severe retinal hemorrhage) was eliminated in the second cohort (9 patients) through improved intraoperative hemostasis. Nonsevere, transient retinal hemorrhages were noted either during or after surgery in all patients as anticipated for a subretinal surgical procedure. Throughout the median 3-year follow-up, results show that implanted eyes were more likely to improve by > 5 letters of BCVA and were less likely to worsen by > 5 letters compared with nonimplanted eyes. CONCLUSIONS: This report details the long-term follow-up of patients with GA to receive a scaffold-based stem cell-derived bioengineered RPE implant. Results show that the implant, at a median 3-year follow-up, is safe and well tolerated in patients with advanced dry age-related macular degeneration. The safety profile, along with the early indication of efficacy, warrants further clinical evaluation of this novel approach for the treatment of GA. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
Asunto(s)
Atrofia Geográfica , Epitelio Pigmentado de la Retina , Agudeza Visual , Humanos , Atrofia Geográfica/cirugía , Atrofia Geográfica/fisiopatología , Epitelio Pigmentado de la Retina/trasplante , Epitelio Pigmentado de la Retina/patología , Anciano , Agudeza Visual/fisiología , Femenino , Anciano de 80 o más Años , Masculino , Estudios de Seguimiento , Tomografía de Coherencia Óptica , Células Madre Embrionarias Humanas/trasplante , Células Madre Embrionarias Humanas/citología , Trasplante de Células Madre , Resultado del TratamientoRESUMEN
ABSTRACT: Retinal pigment epithelium (RPE) transplants rescue photoreceptors in selected animal models of retinal degenerative disease. Early clinical studies of RPE transplants as treatment for age-related macular degeneration (AMD) included autologous and allogeneic transplants of RPE suspensions and RPE sheets for atrophic and neovascular complications of AMD. Subsequent studies explored autologous RPE-Bruch membrane-choroid transplants in patients with neovascular AMD with occasional marked visual benefit, which establishes a rationale for RPE transplants in late-stage AMD. More recent work has involved transplantation of autologous and allogeneic stem cell-derived RPE for patients with AMD and those with Stargardt disease. These early-stage clinical trials have employed RPE suspensions and RPE monolayers on biocompatible scaffolds. Safety has been well documented, but evidence of efficacy is variable. Current research involves development of better scaffolds, improved modulation of immune surveillance, and modification of the extracellular milieu to improve RPE survival and integration with host retina.
Asunto(s)
Epitelio Pigmentado de la Retina , Degeneración Macular Húmeda , Inhibidores de la Angiogénesis , Animales , Humanos , Epitelio Pigmentado de la Retina/trasplante , Suspensiones , Factor A de Crecimiento Endotelial Vascular , Agudeza Visual , Degeneración Macular Húmeda/cirugíaRESUMEN
For many degenerative retinal diseases that progressively lead to blindness, no treatment options are available so far. In recent years, several innovative therapies have been experimentally explored, which are promising because they are independent of the genetic cause of the degenerative disease. One of these is optogenetics, which involves light-sensitive proteins that selectively act as ion channels or ion pumps to control the potential of the treated cell. Thus, these cells can be stimulated or inhibited by light, quasi functionally remote controlled. In this way artificial photoreceptors are induced from the remaining cells, which has already been successfully employed in animal experiments. This type of treatment is already being tested on patients and leads to an improvement in vision, but so far only data from one patient are available. The use of optogenetics additionally requires special eyeglasses to adapt the light impulses in adequate strength and wavelength for the respective optogenes. Another exciting approach is cell replacement therapy of retinal pigment epithelium (RPE) and photoreceptor cells to exchange degenerated cell material. This appears to be very successful for RPE cells in clinical trials. Obtaining human photoreceptors from stem cells is technically possible, but very laborious. The integration of the transplanted photoreceptors into the host retinal tissue also needs further optimization for broader clinical applications; however, both cell replacement and optogenetics approaches are promising, so that the translation from basic research into clinical application will be successful.
Asunto(s)
Oftalmología , Enfermedades de la Retina , Animales , Humanos , Optogenética , Retina , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/trasplanteRESUMEN
BACKGROUND: To observe the ultrastructural outcomes of autologous transplantation of retinal pigment epithelium-partial-thickness choroidal (RPE-PTC) sheets in rabbits after 6 months. METHODS: Eighteen pigmented rabbits were used in this study. Among them, nine rabbits were used for autologous transplantation of RPE-PTC sheets. Tissue sections were observed under a transmission electron microscope for one, three, and six months after transplantation, respectively. RESULTS: One, three, and six months after the autologous transplantation of RPE-PTC sheets, the inner and outer segments of photoreceptor cells were arranged regularly, and the connection between the inner and outer segments was normal. The inner structure of the RPE cells and tight junctions among them remained normal. Phagocytosis of outer segment of photoreceptor cells could also be observed in RPE cells. The structure of the Bruch's membrane appeared loose, rather than being dense as normal, and it was undulated after one and three months, while it became dense after six months. The graft and the bed were healed well, the boundary was unclear, and the graft was vascularized after one, three, and six months, respectively. CONCLUSIONS: Our findings revealed that the RPE-PTC sheets could quickly rebuild blood vessels, thereby maintaining the normal physiological functions of RPE cells, as well as the survival and functional status of photoreceptor cells for a long-time.
Asunto(s)
Coroides , Epitelio Pigmentado de la Retina , Animales , Lámina Basal de la Coroides/trasplante , Lámina Basal de la Coroides/ultraestructura , Coroides/trasplante , Coroides/ultraestructura , Conejos , Epitelio Pigmentado de la Retina/trasplante , Trasplante AutólogoRESUMEN
BACKGROUND: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch's membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes. METHODS: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch's-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome. RESULTS: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation. CONCLUSIONS: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.
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Lámina Basal de la Coroides , Nanofibras , Péptidos , Epitelio Pigmentado de la Retina/trasplante , Ingeniería de Tejidos , Materiales Biocompatibles , Biomimética/métodos , Adhesión Celular , Trasplante de Células , Células Cultivadas , Fenómenos Químicos , Humanos , Integrinas/metabolismo , Nanofibras/química , Nanofibras/ultraestructura , Péptidos/metabolismo , Análisis EspectralRESUMEN
Regenerative medicine relies on basic research outcomes that are only practical when cost effective. The human eyeball requires the retinal pigment epithelium (RPE) to interface the neural retina and the choroid at large. Millions of people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among RPE degradation pathologies. Recently, autologous pluripotent stem-cell-derived RPE cells were prohibitively expensive due to time; therefore, we developed a faster reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts (Fibs) by conditional overexpression of both broad plasticity and lineage-specific transcription factors (TFs). iRPE cells displayed critical RPE benchmarks and significant in vivo integration in transplanted retinas. Herein, we detail the iRPE system with comprehensive single-cell RNA sequencing (scRNA-seq) profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for basic research and affordable autologous human RPE tissue for regenerative cell therapy.
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Reprogramación Celular , Fibroblastos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Reprogramación Celular/efectos de los fármacos , Disulfuros/farmacología , Fibroblastos/citología , Regulación de la Expresión Génica , Humanos , Alcaloides Indólicos/farmacología , Aprendizaje Automático , Niacinamida/farmacología , Ratas , Retina/citología , Retina/metabolismo , Retina/patología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/trasplante , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The main cause of progressive vision impairment in retinal degenerative diseases is the dysfunction of photoreceptors and the underlying retinal pigment epithelial cells. The inadequate regenerative capacity of the neural retina and lack of established therapeutic options demand the development of clinical-grade protocols to halt the degenerative process in the eye or replace the damaged cells by using stem cell-derived products. Recently, stem cell-based regenerative therapies have been at the forefront of clinical investigations for retinal dystrophies. OBJECTIVE: This article will review different stem cell-based therapies currently employed for retinal degenerative diseases, recent clinical trials, and major challenges in the translation of these therapies from bench to bedside. METHODOLOGY: A systematic literature review was conducted to identify potentially relevant articles published in MEDLINE/PubMed, Embase, ClinicalTrials.gov, Drugs@FDA, European Medicines Agency, and World Health Organization International Clinical Trials Registry Platform. RESULTS: Transplantation of healthy cells to replace damaged cells in the outer retina is a clinically relevant concept because the inner retina that communicates with the visual areas of the brain remains functional even after the photoreceptors are completely lost. Various methods have been established for the differentiation of pluripotent stem cells into different retinal cell types that can be used for therapies. Factors released from transplanted somatic stem cells showed trophic support and photoreceptor rescue during the early stages of the disease. Several preclinical and phase I/II clinical studies using terminally differentiated photoreceptor/retinal pigment epithelial cells derived from pluripotent stem cells have shown proof of concept for visual restoration in Age-related Macular Degeneration (AMD), Stargardt disease, and Retinitis Pigmentosa (RP). CONCLUSION: Cell replacement therapy has great potential for vision restoration. The results obtained from the initial clinical trials are encouraging and indicate its therapeutic benefits. The current status of the therapies suggests that there is a long way to go before these results can be applied to routine clinical practice. Input from the ongoing multicentre clinical trials will give a more refined idea for the future design of clinical-grade protocols to transplant GMP level HLA matched cells.
Asunto(s)
Células Madre Pluripotentes , Degeneración Retiniana , Humanos , Retina , Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/trasplante , Pigmentos Retinianos , Trasplante de Células Madre/métodosRESUMEN
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch's membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
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Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/trasplante , Ingeniería de Tejidos/métodos , Adhesión Celular , Línea Celular , Células Cultivadas , Coroides/citología , Proteínas del Ojo/metabolismo , Células Madre Embrionarias Humanas , Humanos , Degeneración Macular/terapia , Factores de Crecimiento Nervioso/metabolismo , Retina/citología , Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Serpinas/metabolismoRESUMEN
Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.
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Células Madre Pluripotentes Inducidas/citología , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Degeneración Retiniana/cirugía , Epitelio Pigmentado de la Retina/trasplante , Animales , Masculino , Conejos , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Degeneración Retiniana/patología , Degeneración Retiniana/terapiaRESUMEN
Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Epitelio Pigmentado de la Retina/trasplante , Animales , Biomarcadores/metabolismo , Humanos , Implantes Experimentales , Luz , Polímeros , Ratas , Colículos Superiores/efectos de la radiación , Análisis de Supervivencia , Visión Ocular/efectos de la radiación , XilenosRESUMEN
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.
Asunto(s)
Atrofia Geográfica , Células Madre Pluripotentes Inducidas , Epitelio Pigmentado de la Retina , Animales , Antígenos de Diferenciación/biosíntesis , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Atrofia Geográfica/metabolismo , Atrofia Geográfica/patología , Atrofia Geográfica/cirugía , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/trasplante , PorcinosRESUMEN
The ocular tissue microenvironment is immune privileged and uses several mechanisms of immunosuppression to prevent the induction of inflammation. Besides being a blood-barrier and source of photoreceptor nutrients, the retinal pigment epithelial cells (RPE) regulate the activity of immune cells within the retina. These mechanisms involve the expression of immunomodulating molecules that make macrophages and microglial cells suppress inflammation and promote immune tolerance. The RPE have an important role in ocular immune privilege to regulate the behavior of immune cells within the retina. Reviewed is the current understanding of how RPE mediate this regulation and the changes seen under pathological conditions.
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Macrófagos/inmunología , Microglía/inmunología , Retina/trasplante , Epitelio Pigmentado de la Retina/trasplante , Animales , Humanos , Tolerancia Inmunológica , Ratones , Retina/inmunología , Epitelio Pigmentado de la Retina/inmunología , Inmunología del Trasplante , Trasplante HomólogoRESUMEN
Replacement of dysfunctional retinal pigmented epithelium (RPE) with grafts derived from stem cells has the potential to improve vision for patients with retinal disorders. In fact, the potential is such that a great number of groups are attempting to realize this therapy through individual strategies with a variety of stem cell products, hosts, immunomodulatory regimen, and techniques to assess the success of their design. Comparing the findings of different investigators is complicated by a number of factors. The immune response varies greatly between xenogeneic and allogeneic transplantation. A unique immunologic environment is created in the subretinal space, the target of RPE grafts. Both functional assessment and imaging techniques used to evaluate transplants are susceptible to erroneous conclusions. Lastly, the pharmacologic regimens used in RPE transplant trials are as numerous and variable as the trials themselves, making it difficult to determine useful results. This review will discuss the causes of these complicating factors, digest the strategies and results from clinical and preclinical studies, and suggest places for improvement in the design of future transplants and investigations.
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Rechazo de Injerto/inmunología , Células Madre Pluripotentes Inducidas/fisiología , Degeneración Macular/terapia , Trasplante de Órganos , Epitelio Pigmentado de la Retina/fisiología , Animales , Humanos , Epitelio Pigmentado de la Retina/trasplante , Tolerancia al TrasplanteRESUMEN
Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.
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Criopreservación/métodos , Células Epiteliales/trasplante , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/trasplante , Manejo de Especímenes/métodos , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Ojo/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , Factores de Crecimiento Nervioso/metabolismo , Polímeros , Ratas , Ratas Desnudas , Medicina Regenerativa/métodos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Serpinas/metabolismo , Andamios del Tejido , Resultado del Tratamiento , XilenosRESUMEN
Progenitor cells derived from the retinal pigment epithelium (RPECs) have shown promise as therapeutic approaches to degenerative retinal disorders including diabetic retinopathy, age-related macular degeneration and Stargardt disease. However, the degeneration of Bruch's membrane (BM), the natural substrate for the RPE, has been identified as one of the major limitations for utilizing RPECs. This degeneration leads to decreased support, survival and integration of the transplanted RPECs. It has been proposed that the generation of organized structures of nanofibers, in an attempt to mimic the natural retinal extracellular matrix (ECM) and its unique characteristics, could be utilized to overcome these limitations. Furthermore, nanoparticles could be incorporated to provide a platform for improved drug delivery and sustained release of molecules over several months to years. In addition, the incorporation of tissue-specific genes and stem cells into the nanostructures increased the stability and enhanced transfection efficiency of gene/drug to the posterior segment of the eye. This review discusses available drug delivery systems and combination therapies together with challenges associated with each approach. As the last step, we discuss the application of nanofibrous scaffolds for the implantation of RPE progenitor cells with the aim to enhance cell adhesion and support a functionally polarized RPE monolayer.
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Portadores de Fármacos/química , Nanofibras/química , Enfermedades de la Retina/terapia , Epitelio Pigmentado de la Retina/trasplante , Trasplante de Células Madre/métodos , Andamios del Tejido/química , Animales , Lámina Basal de la Coroides/química , Retinopatía Diabética/terapia , Sistemas de Liberación de Medicamentos/métodos , Humanos , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/citología , Enfermedad de Stargardt/terapia , Células Madre/citologíaRESUMEN
Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.
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Criopreservación/métodos , Epitelio Pigmentado de la Retina/metabolismo , Trasplante de Células Madre/métodos , Membrana Basal/metabolismo , Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular , Colágeno Tipo IV/metabolismo , Humanos , Laminina/metabolismo , Degeneración Macular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fagocitosis/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Epitelio Pigmentado de la Retina/trasplante , Manejo de Especímenes/métodosRESUMEN
Recent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.
Asunto(s)
Xenoinjertos/trasplante , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/trasplante , Trasplante de Células Madre/métodos , Anciano , Anciano de 80 o más Años , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Xenoinjertos/patología , Humanos , Terapia de Inmunosupresión , Macaca fascicularis , Masculino , Células Fotorreceptoras/fisiología , Primates , Retina/patología , Retina/trasplante , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.