Single -cell transcriptomic profiling of stem cell derived retinal pigment epithelial cell monolayers after subretinal transplantation
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ABSTRACT:
PROVIDER: PRJNA877840 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21484600.fastq.gz | Fastqsanger.gz | |||
| SRR21484601.fastq.gz | Fastqsanger.gz | |||
| SRR21484602.fastq.gz | Fastqsanger.gz | |||
| SRR21484603.fastq.gz | Fastqsanger.gz | |||
| SRR21484604.fastq.gz | Fastqsanger.gz |
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Project description:Transplantation of stem cell derived retinal pigment epithelial (RPE) cells can potentially restore vision in patients with age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation and fate specification of transplanted RPE cells. To address this, we transplanted stem cell derived RPE into the subretinal space of immunocompetent rabbits for one month and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation trajectory towards the native adult human RPE state in all transplanted RPE cells, regardless of stem cell resource. Using adult human RPE cells as a reference, we reconstructed the gene regulatory networks in our transplanted stem cell derived RPE, and identified tripartite transcription factors (MAFF, JUND and FOS), known to modulate a broad array of RPE functions in vivo. This includes regulation of canonical RPE signature genes known to support host photoreceptors, and pro-survival genes required for adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.
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Project description:Injuries to the retinal pigment epithelium (RPE) and outer retina often result in the accumulation of retinal microglia within the subretinal space. These subretinal microglia play crucial roles in inflammation and resolution, but the mechanisms governing their functions are still largely unknown. Our previous research highlighted the protective functions of choroidal gd T cells in response to RPE injury. In the current study, we employed single-cell RNA sequencing approach to delve deeper into the mechanisms involved. We found that gd T cells were the primary producer of interleukin-17 (IL-17) in the choroid. IL-17 signaled through its receptor on the RPE, subsequently triggering the production of interleukin-6 (IL-6). This cascade of cytokines initiated a metabolic reprogramming of subretinal microglia, enhancing their capacity for lipid metabolism. RPE-specific knockout of IL-17 receptor A led to the dysfunction of subretinal microglia and RPE pathology. Collectively, our findings suggest that responding to RPE injury, the choroidal gd T cells can initiate a protective signaling cascade that ensures the proper functioning of subretinal microglia.
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Project description:Subretinal fibrosis permanently impairs the vision of patients with neovascular age-related macular degeneration. Despite emerging evidence revealing the association between disturbed metabolism in retinal pigment epithelium (RPE) and subretinal fibrosis, the underlying mechanism remains unclear. In the present study, single-cell RNA sequencing revealed, prior to subretinal fibrosis, genes in mitochondrial fatty acid oxidation are downregulated in the RPE lacking very low-density lipoprotein receptor (VLDLR), especially the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A). We found that overexpression of CPT1A in the RPE of Vldlr-/- mice suppresses epithelial-to-mesenchymal transition and fibrosis. Mechanistically, TGFβ2 induces fibrosis by activating a Warburg-like effect, i.e. increased glycolysis and decreased mitochondrial respiration through ERK-dependent CPT1A degradation. Moreover, VLDLR blocks the formation of the TGFβ receptor I/II complex by interacting with unglycosylated TGFβ receptor II. In conclusion, VLDLR suppresses fibrosis by attenuating TGFβ2-induced metabolic reprogramming, and CPT1A is a potential target for treating subretinal fibrosis.
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Project description:Age-related macular degeneration (AMD), featured with dysfunction and loss of retinal pigment epithelium (RPE), is lacking efficient therapeutic approaches. According to our previous studies, human amniotic epithelial stem cells (hAESCs) may serve as a potential seed cell source of RPE cells for therapy because they have no ethical concerns, no tumorigenicity, and little immunogenicity. Herein, trichostatin A and nicotinamide can direct hAESCs differentiation into RPE like cells. The differentiated cells display the morphology, marker expression and cellular function of the native RPE cells, and noticeably express little MHC class II antigens and high level of HLA-G. Importantly, visual function and retinal structure of Royal College of Surgeon (RCS) rats, a classical animal model of retinal degeneration, were rescued after subretinal transplantation with the hAESCs-derived RPE like cells. We established a high-efficient, low-cost and safety-guaranteed system for generating functional RPE cells from hAESCs. These results suggest a novel and ideal therapeutic strategy for retinal degeneration diseases
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Project description:In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.
| S-EPMC4659637 | biostudies-literature
Project description:Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here we conduct single-cell transcriptomic profiling of a hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, in which cells rapidly acquired a rostral embryo patterning signature, before converging towards the RPE lineage. At intermediate steps, we identified and examined the potency of a NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.
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Project description:This is the proteomic analysis data of bovine retinal pigment epithelium cell lysate subjected to cryo-EM study.
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Project description:Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF–β) and the inflammatory cytokine tumor necrosis factor alpha (TNF–α), can induce RPE–EMT; however, small molecule inhibitors of RPE–EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF–β/TNF–α-induced RPE–EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE–EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE–EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.
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Project description:In many forms of retinal degenerative diseases in human, microglia relocate to and accumulate in the subretinal space. However, the roles of microglia in retinal degeneration are poorly understood. By leveraging single cell RNA-seq, we identified a distinct microglia subtype in the subretinal space. These microglia underwent transcriptional reprogramming characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. Importantly, this transition is associated with protection of the retinal pigment epithelium from damage caused by disease. Therefore, our data demonstrated microglial heterogeneity in retinal degeneration and may provide important implications for developing new strategies to prevent loss of vision.
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