Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells ...have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent ...and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Degeneration of the retinal pigment epithelium (RPE) plays a central role in age-related macular degeneration (AMD). Throughout life, RPE cells are challenged by a variety of cytotoxic stressors, ...some of which are cumulative with age and may ultimately contribute to drusen and lipofuscin accumulation. Stressors such as these continually damage RPE cells resulting in a state of chronic wounding. Current cell-based platforms that model a state of chronic RPE cell wounding are limited, and the RPE cellular response is not entirely understood. Here, we used the electric cell-substrate impedance sensing (ECIS) system to induce a state of acute or chronic wounding on differentiated human fetal RPE cells to analyze changes in the wound repair response. RPE cells surrounding the lesioned area employ both cell migration and proliferation to repair wounds but fail to reestablish their original cell morphology or density after repetitive wounding. Chronically wounded RPE cells develop phenotypic AMD characteristics such as loss of cuboidal morphology, enlarged size, and multinucleation. Transcriptomic analysis suggests a systemic misregulation of RPE cell functions in bystander cells, which are not directly adjacent to the wound. Genes associated with the major RPE cell functions (LRAT, MITF, RDH11) significantly downregulate after wounding, in addition to differential expression of genes associated with the cell cycle (CDK1, CDC6, CDC20), inflammation (IL-18, CCL2), and apoptosis (FAS). Interestingly, repetitive wounding resulted in prolonged misregulation of genes, including FAS, LRAT, and PEDF. The use of ECIS to induce wounding resulted in an over-representation of AMD-associated genes among those dysregulated genes, particularly genes associated with advanced AMD. This simple system provides a new model for further investigation of RPE cell wound response in AMD pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The nuclear factor one (NFI) transcription factor genes
,
, and
are all enriched in late-stage retinal progenitor cells, and their loss has been shown to retain these progenitors at the expense of ...later-generated retinal cell types. Whether they play any role in the specification of those later-generated fates is unknown, but the expression of one of these,
, in a specific amacrine cell type may intimate such a role. Here,
conditional knockout (
-CKO) mice (both sexes) were assessed, finding a massive and largely selective absence of AII amacrine cells. There was, however, a partial reduction in type 2 cone bipolar cells (CBCs), being richly interconnected to AII cells. Counts of dying cells showed a significant increase in
-CKO retinas at postnatal day (P)7, after AII cell numbers were already reduced but in advance of the loss of type 2 CBCs detected by P10. Those results suggest a role for
in the specification of the AII amacrine cell fate and a dependency of the type 2 CBCs on them. Delaying the conditional loss of
to the first postnatal week did not alter AII cell number nor differentiation, further suggesting that its role in AII cells is solely associated with their production. The physiological consequences of their loss were assessed using the ERG, finding the oscillatory potentials to be profoundly diminished. A slight reduction in the b-wave was also detected, attributed to an altered distribution of the terminals of rod bipolar cells, implicating a role of the AII amacrine cells in constraining their stratification.
The transcription factor NFIA is shown to play a critical role in the specification of a single type of retinal amacrine cell, the AII cell. Using an
-conditional knockout mouse to eliminate this population of retinal neurons, we demonstrate two selective bipolar cell dependencies on the AII cells; the terminals of rod bipolar cells become mis-stratified in the inner plexiform layer, and one type of cone bipolar cell undergoes enhanced cell death. The physiological consequence of this loss of the AII cells was also assessed, finding the cells to be a major contributor to the oscillatory potentials in the electroretinogram.
The innate capacity of adult somatic cells has many potential applications in regenerative medicine. In this issue of Cell Stem Cell, Salero et al. (2012) describe an adult retinal stem cell ...population capable of generating neural and mesenchymal cell lineages.
Induced pluripotent stem cells (iPSCs) have the potential to transform drug discovery and healthcare in the 21st century. However, successful commercialization will require standardized manufacturing ...platforms. Here we highlight the need to define standardized practices for iPSC generation and processing and discuss current challenges to the robust manufacture of iPSC products.
Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of ...neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD.
Transcriptome wide expression profiles of human fetal RPE cell cultures as a function of passage and time post-plating were determined using Agilent 44 K whole genome microarrays and RNA-Seq. Using a systems level analysis, differentially expressed genes and pathways of interest were identified and their role in the establishment of a persistent mesenchymal state was assessed using pharmacological-based experiments.
Using a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we show that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40 % of the transcriptome. In contrast, at subconfluence fewer than 5 % of expressed transcripts have two-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFβ pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and largely reverses the passage-dependent loss of epithelial potential; thus extending the effective lifespan by at least four passages. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD, including key members of the TGFβ pathway.
In RPE cells the switch to a persistent mesenchymal state following prolonged wound stimulus is driven by lasting activation of the TGFβ pathway. Targeted inhibition of TGFβ signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell-based therapies.
Retinal pigment epithelial (RPE) transplantation aims to restore the subretinal anatomy and re-establish the critical interaction between the RPE and the photoreceptor, which is fundamental to sight. ...The field has developed over the past 20 years with advances coming from a large body of animal work and more recently a considerable number of human trials. Enormous progress has been made with the potential for at least partial restoration of visual function in both animal and human clinical work. Diseases that have been treated with RPE transplantation demonstrating partial reversal of vision loss include primary RPE dystrophies such as the merTK dystrophy in the Royal College of Surgeons (RCS) rat and in humans, photoreceptor dystrophies as well as complex retinal diseases such as atrophic and neovascular age-related macular degeneration (AMD). Unfortunately, in the human trials the visual recovery has been limited at best and full visual recovery has not been demonstrated. Autologous full-thickness transplants have been used most commonly and effectively in human disease but the search for a cell source to replace autologous RPE such as embryonic stem cells, marrow-derived stem cells, umbilical cord-derived cells as well as immortalised cell lines continues. The combination of cell transplantation with other modalities of treatment such as gene transfer remains an exciting future prospect. RPE transplantation has already been shown to be capable of restoring the subretinal anatomy and improving photoreceptor function in a variety of retinal diseases. In the near future, refinements of current techniques are likely to allow RPE transplantation to enter the mainstream of retinal therapy at a time when the treatment of previously blinding retinal diseases is finally becoming a reality.
Transforming growth factor β-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelium (RPE) ...differentiation. We have shown previously that inhibition of TGFβ signaling restored the capacity of mesenchymal RPE to differentiate; however, the potential lessens with extensive passaging. We investigated TGFβ-independent mechanisms that regulate RPE differentiation following repetitive passaging.
Retinal pigment epithelium-EMT was induced by repetitive passaging of fetal RPE at subconfluence. Suppression of EMT was achieved by the addition of A-83-01, a TGFβ receptor kinase inhibitor. Transcriptomic analysis was used to identify potential TGFβ independent processes that prevent differentiation after extensive passage. Downregulated transcription factors were identified and transduced into highly passaged RPE to restore cell differentiation. Restoration was evaluated according to morphology, expression of RPE/mesenchymal markers, transcriptomic analysis, cell doubling time, and senescence-associated β-galactosidase (SA-β-gal) activity.
A-83-01-treated RPE failed to differentiate after 7 passages (P7). This failure was concomitant with downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential, and cell senescence. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 restored P7-RPE differentiation to a status similar to minimally passaged RPE. Moreover, the treatment allowed cells to maintain their differentiation capacity after extended passaging.
Retinal pigment epithelium subjected to chronic wound stimulus undergoes TGFβ-mediated EMT, loss of expression of signature RPE genes, and senescence. Targeting these aspects with a TGFβ receptor kinase inhibitor, a RPE transcription factor, and a cell cycle regulator restores the capacity of highly passaged RPE cells to regenerate and differentiate.
Glaucoma is one of the leading causes of blindness, and there is an ongoing need for new therapies. Recent studies indicate that cell transplantation using Müller glia may be beneficial, but there is ...a need for novel sources of cells to provide therapeutic benefit. In this study, we have isolated Müller glia from retinal organoids formed by human induced pluripotent stem cells (hiPSCs) in vitro and have shown their ability to partially restore visual function in rats depleted of retinal ganglion cells by NMDA. Based on the present results, we suggest that Müller glia derived from retinal organoids formed by hiPSC may provide an attractive source of cells for human retinal therapies, to prevent and treat vision loss caused by retinal degenerative conditions. Stem Cells Translational Medicine 2019;8:775&784
Purified populations of Müller glial cells were isolated from retinal organoids derived from human iPSC. Intra‐vitreal transplantation of these cells into rat eyes depleted of RGC by NMDA caused partial restoration of visual function, as assessed by improvement of the retinal electrophysiological responses. These findings highlight the potential of iPSC as a potential source of therapeutic cells for retinal degeneration.
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