Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Single cell RNAseq of 12-week old AKIMBA versus WT mouse retina

ABSTRACT: Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2AkitaxVEGF+/-) mice, which are known to replicate features of clinical diabetic retinopathy. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by measuring the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. Retinas (n=4 for Akimba, n=2 for wild-type) were isolated in ice-cold Dulbecco’s Modified Eagle Medium. After rinsing with Dulbecco’s Phosphate-Buffered Saline containing 2% fetal bovine serum, each retina was incubated with 1mL digestion buffer (2mg/mL collagenase-P, 200U/mL DNAse-I (Sigma-Aldrich) in M199 medium (Life Technologies) at 37°C for 10min. Retinal tissue was further dissociated by trituration and the suspension was filtered through a 40µm cell strainer and centrifuged for 5min at 300xg (4°C). Pooled retinal single-cell suspensions from wild-type and Akimba were counted on a Luna-FL Cell Counter (Logos Biosystems) and libraries were prepared with the Chromium Single-cell 3’ V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics) aiming for 5000 cells per library. Barcoded libraries were sequenced on an Illumina HiSeq4000 in 25-8-98 paired-end configuration. The transcriptome data for 9474 retinal cells were analysed, yielding 15 clusters corresponding to eight distinct retinal cell types.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Michael Porcu

PROVIDER: E-MTAB-9061 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy.

Van Hove Inge I De Groef Lies L Boeckx Bram B Modave Elodie E Hu Tjing-Tjing TT Beets Karen K Etienne Isabelle I Van Bergen Tine T Lambrechts Diether D Moons Lieve L Feyen Jean H M JHM Porcu Michaël M

Diabetologia 20200730 10

<h4>Aims/hypothesis</h4>Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabet ...[more]

PMID: 32734440

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Project description:Human CD34+ stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34+ stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34+ BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34+ cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34+ BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34+ BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34+ BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34+ BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34+ BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34+ BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

Dataset Information

Single cell RNAseq of 12-week old AKIMBA versus WT mouse retina

Publications

Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy.

Similar Datasets

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