ORCID Profile
0000-0002-1818-0375
Current Organisation
Children's Medical Research Institute
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Publisher: Elsevier BV
Date: 02-2018
Publisher: Cold Spring Harbor Laboratory
Date: 17-05-2021
DOI: 10.1101/2021.05.16.444356
Abstract: Advances in the study of neurological conditions have been possible due to induced pluripotent stem cell technologies and the generation of neural cell types and organoids. Numerous studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and organoid-to-organoid variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids were isolated and cultured in suspension conditions for maturation and are transcriptionally comparable to organoids generated by other methods and to human foetal cortex. Furthermore, these organoids show spontaneous functional network activity with proteomic analysis and electron microscopy demonstrating the presence of synaptic components and maturity. The generation of retinal and brain organoids in close proximity also enabled their mutual isolation. Further culture of this complex organoid system demonstrated the formation of optic nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of the mechanisms of neurological diseases of the eye and brain.
Publisher: Mary Ann Liebert Inc
Date: 05-2020
DOI: 10.1089/HUM.2019.264
Publisher: Elsevier BV
Date: 2023
Publisher: Springer Science and Business Media LLC
Date: 13-06-2018
Publisher: Springer Science and Business Media LLC
Date: 21-07-2013
DOI: 10.1038/NBT.2643
Publisher: Elsevier BV
Date: 06-2022
Publisher: The Company of Biologists
Date: 2016
DOI: 10.1242/DMM.022475
Abstract: Endotoxin-induced uveitis (EIU) is a model of acute TLR4-mediated organ inflammation, which has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific cell count scoring with histological sections upon each eye as a measure of disease severity. Here we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate in Ccl2- and Ccr2-deficient mice. Optical coherence tomography (OCT) was used for the first time in this model to allow in vivo imaging of infiltrating vitreous cells and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model.
Publisher: Mary Ann Liebert Inc
Date: 06-2022
DOI: 10.1089/HUM.2021.278
Abstract: The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an
Publisher: Oxford University Press (OUP)
Date: 27-05-2015
DOI: 10.1002/STEM.2051
Abstract: Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application. Stem Cells 2015 :2469—2482
Publisher: SAGE Publications
Date: 05-2012
Abstract: 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.
Publisher: Elsevier
Date: 2017
DOI: 10.1016/BS.PBR.2017.01.001
Abstract: Age-related macular degeneration and inherited retinal degenerations represent the leading causes of blindness in industrialized countries. Despite different initiating causes, they share a common final pathophysiology, the loss of the light sensitive photoreceptors. Replacement by transplantation may offer a potential treatment strategy for both patient populations. The last decade has seen remarkable progress in our ability to generate retinal cell types, including photoreceptors, from a variety of murine and human pluripotent stem cell sources. Driven in large part by the requirement for renewable cell sources, stem cells have emerged not only as a promising source of replacement photoreceptors but also to provide in vitro systems with which to study retinal development and disease processes and to test therapeutic agents.
Publisher: The Company of Biologists
Date: 11-2022
DOI: 10.1242/DMM.049593
Abstract: The sensory epithelia of the mammalian inner ear enable sound and movement to be perceived. Damage to these epithelia can cause irreversible sensorineural hearing loss and vestibular dysfunction because they lack regenerative capacity. The human inner ear cannot be biopsied without causing permanent damage, significantly limiting the tissue s les available for research. Investigating disease pathology and therapeutic developments have therefore traditionally relied on animal models, which often cannot completely recapitulate the human otic systems. These challenges are now being partly addressed using induced pluripotent stem cell-derived cultures, which generate the sensory epithelial-like tissues of the inner ear. Here, we review how pluripotent stem cells have been used to produce two-dimensional and three-dimensional otic cultures, the strengths and limitations of these new approaches, and how they have been employed to investigate genetic and acquired forms of audiovestibular dysfunction. This Review provides an overview of the progress in pluripotent stem cell-derived otic cultures thus far, focusing on their applications in disease modelling and therapeutic trials. We survey their current limitations and future directions, highlighting their prospective utility for high-throughput drug screening and developing personalised medicine approaches.
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.PRETEYERES.2015.01.003
Abstract: Loss of photoreceptors due to retinal degeneration is a major cause of blindness in the developed world. While no effective treatment is currently available, cell replacement therapy, using pluripotent stem cell-derived photoreceptor precursor cells, may be a feasible future treatment. Recent reports have demonstrated rescue of visual function following the transplantation of immature photoreceptors and we have seen major advances in our ability to generate transplantation-competent donor cells from stem cell sources. Moreover, we are beginning to realise the possibilities of using endogenous populations of cells from within the retina itself to mediate retinal repair. Here, we present a review of our current understanding of endogenous repair mechanisms together with recent progress in the use of both ocular and pluripotent stem cells for the treatment of photoreceptor loss. We consider how our understanding of retinal development has underpinned many of the recent major advances in translation and moved us closer to the goal of restoring vision by cellular means.
Publisher: Elsevier BV
Date: 09-2017
Publisher: Elsevier BV
Date: 04-2022
Publisher: Oxford University Press (OUP)
Date: 18-06-2012
DOI: 10.1002/STEM.1123
Abstract: Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), 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 ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-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 ESC-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 ESC-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.
Publisher: Elsevier BV
Date: 06-2017
Publisher: Mary Ann Liebert Inc
Date: 10-2018
DOI: 10.1089/HUM.2018.027
Abstract: Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.
Publisher: The Company of Biologists
Date: 15-12-2020
DOI: 10.1242/DEV.189746
Abstract: Retinal development and maturation are orchestrated by a series of interacting signalling networks that drive the morphogenetic transformation of the anterior developing brain. Studies in model organisms continue to elucidate these complex series of events. However, the human retina shows many differences from that of other organisms and the investigation of human eye development now benefits from stem cell-derived organoids. Retinal differentiation methods have progressed from simple 2D adherent cultures to self-organising micro-physiological systems. As models of development, these have collectively offered new insights into the previously unexplored early development of the human retina and informed our knowledge of the key cell fate decisions that govern the specification of light-sensitive photoreceptors. Although the developmental trajectories of other retinal cell types remain more elusive, the collation of omics datasets, combined with advanced culture methodology, will enable modelling of the intricate process of human retinogenesis and retinal disease in vitro.
Publisher: MDPI AG
Date: 21-03-2022
DOI: 10.3390/JPM12030502
Abstract: The RPGR gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in RPGR are the main contributor to X-linked rod-cone dystrophy (RCD), and RPGR gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel RPGR variants is important for a patient therapy opportunity. Here, we describe a novel intronic RPGR variant, c.1415 − 9A G, in a patient with RCD, which was classified as a variant of uncertain significance according to current clinical diagnostic criteria. The variant lay several base pairs intronic to the canonical splice acceptor site, raising suspicion of an RPGR RNA splicing abnormality and consequent protein dysfunction. To investigate disease causation in an appropriate disease model, induced pluripotent stem cells were generated from patient fibroblasts and differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO). Abnormal RNA splicing of RPGR was demonstrated in patient fibroblasts, iPSC-RPE and iPSC-ROs, leading to a predicted frameshift and premature stop codon. Decreased RPGR expression was demonstrated in these cell types, with a striking loss of RPGR localization at the ciliary transitional zone, critically in the photoreceptor cilium of the patient iPSC-ROs. Mislocalisation of rhodopsin staining was present in the patient’s iPSC-RO rod photoreceptor cells, along with an abnormality of L/M opsin staining affecting cone photoreceptor cells and increased photoreceptor apoptosis. Additionally, patient iPSC-ROs displayed an increase in F-actin expression that was consistent with an abnormal actin regulation phenotype. Collectively, these studies indicate that the splicing abnormality caused by the c.1415 − 9A G variant has an impact on RPGR function. This work has enabled the reclassification of this variant to pathogenic, allowing the consideration of patients with this variant having access to gene therapy clinical trials. In addition, we have identified biomarkers of disease suitable for the interrogation of other RPGR variants of uncertain significance.
Publisher: The Company of Biologists
Date: 2020
DOI: 10.1242/DEV.188730
Abstract: Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub dm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.
Publisher: Elsevier BV
Date: 04-2021
Publisher: Springer Science and Business Media LLC
Date: 25-07-2018
Publisher: The Company of Biologists
Date: 15-02-2014
DOI: 10.1242/DEV.104901
Abstract: Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of in idual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Springer Science and Business Media LLC
Date: 07-11-2017
DOI: 10.1038/S41598-017-15212-Z
Abstract: Age-related macular degeneration (AMD) is a major cause of blindness and is associated with complement dysregulation. The disease is a potential target for stem cell therapy but success is likely to be limited by the inflammatory response. We investigated the innate immune properties of human induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression of CL-11 in iPS-RPE cells, and in the extracellular fluid. Complement activation on the cell surface occurred in conjunction with CL-11 binding. CL-11 has been shown to activate inflammatory responses through recognition of L-fucose, which we confirmed by showing that fucosidase-treated cells, largely, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions.
Publisher: Oxford University Press (OUP)
Date: 19-08-2011
DOI: 10.1002/STEM.694
Abstract: 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 donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Anai Gonzalez Cordero.