scholarly journals Single cell RNA sequencing of stem cell-derived retinal ganglion cells

2018 ◽  
Vol 5 (1) ◽  
Author(s):  
Maciej Daniszewski ◽  
Anne Senabouth ◽  
Quan H. Nguyen ◽  
Duncan E. Crombie ◽  
Samuel W. Lukowski ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Retinal Ganglion ◽  
Single Cell Rna Sequencing

scholarly journals Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells

2017 ◽  
Author(s):  
Maciej Daniszewski ◽  
Anne Senabouth ◽  
Quan Nguyen ◽  
Duncan E. Crombie ◽  
Samuel W. Lukowski ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Retinal Ganglion Cells ◽  
Human Embryonic Stem Cell ◽  
Ganglion Cells ◽  
Embryonic Stem ◽  
Retinal Ganglion ◽  
Single Cell Rna Sequencing

ABSTRACTWe used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and subsequently prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells upregulated genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature phenotype.

scholarly journals Laser capture microdissection-based single-cell RNA sequencing reveals optic nerve crush-related early mRNA alterations in retinal ganglion cells

2020 ◽  
Author(s):  
Dongyan Pan ◽  
Mengqiao Xu ◽  
Xin Chang ◽  
Mao Xia ◽  
Yibin Fang ◽  
...  
Keyword(s):  
Optic Nerve ◽  
Single Cell ◽  
Rna Sequencing ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Optic Nerve Crush ◽  
Retinal Ganglion ◽  
Nerve Crush ◽  
Single Cell Rna Sequencing ◽  
Laser Capture

AbstractRetinal ganglion cells (RGC) are the primary cell type injured in a variety of diseases of the optic nerve, and the early changes of RGC’s RNA profiling may be important to understand the mechanism of optic nerve injury and axon regeneration. Here we employed the optic nerve crush (ONC) model to explore early mRNA alterations in RGCs using laser capture microdissection (LCM) and single-cell RNA sequencing. We successfully established an optimal LCM protocol using 30 μm-thick retinal tissue sections mounted on glass slides and laser pressure catapulting (LPC) to collect RGCs and obtain high-quality RNA for single-cell sequencing. Based on our protocol, we identified 8744 differentially expressed genes that were involved in ONC-related early mRNA alterations in RGCs. Candidate genes included Atf3, Lgals3, LOC102551701, Plaur, Tmem140 and Maml1. The LCM-based single-cell RNA sequencing allowed new insights into the early mRNA changes in RGCs, highlighting new molecules associated with ONC.

scholarly journals Single Cell Sequencing of Induced Pluripotent Stem Cell Derived Retinal Ganglion Cells (iPSC-RGC) Reveals Distinct Molecular Signatures and RGC Subtypes

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 2015
Author(s):  
Harini V. Gudiseva ◽  
Vrathasha Vrathasha ◽  
Jie He ◽  
Devesh Bungatavula ◽  
Joan M. O’Brien ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Single Cell ◽  
Retinal Ganglion Cells ◽  
Pluripotent Stem Cell ◽  
Ganglion Cells ◽  
Induced Pluripotent Stem Cell ◽  
Retinal Ganglion ◽  
Single Cell Sequencing ◽  
Induced Pluripotent

We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3’ V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.

scholarly journals Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches

Genes ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 112
Author(s):  
Marta García-López ◽  
Joaquín Arenas ◽  
M. Esther Gallardo
Keyword(s):  
Stem Cell ◽  
Retinal Ganglion Cells ◽  
Pluripotent Stem Cell ◽  
Ganglion Cells ◽  
Genetic Disorders ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Mechanism ◽  
Retinal Ganglion ◽  
Induced Pluripotent

Inherited optic neuropathies share visual impairment due to the degeneration of retinal ganglion cells (RGCs) as the hallmark of the disease. This group of genetic disorders are caused by mutations in nuclear genes or in the mitochondrial DNA (mtDNA). An impaired mitochondrial function is the underlying mechanism of these diseases. Currently, optic neuropathies lack an effective treatment, and the implementation of induced pluripotent stem cell (iPSC) technology would entail a huge step forward. The generation of iPSC-derived RGCs would allow faithfully modeling these disorders, and these RGCs would represent an appealing platform for drug screening as well, paving the way for a proper therapy. Here, we review the ongoing two-dimensional (2D) and three-dimensional (3D) approaches based on iPSCs and their applications, taking into account the more innovative technologies, which include tissue engineering or microfluidics.

scholarly journals 973. Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Cerebral Organoids

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S33-S34
Author(s):  
Karen Ocwieja ◽  
Alexandra Stanton ◽  
Alexsia Richards ◽  
Jenna Antonucci ◽  
Travis Hughes ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Brain ◽  
Single Cell ◽  
Rna Sequencing ◽  
Zika Virus ◽  
Model Systems ◽  
Sequencing Analysis ◽  
Human Stem Cell ◽  
Zikv Infection ◽  
Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. One barrier has been the lack of relevant model systems for the developing human brain; however, thanks to advances in the stem cell field, we can now evaluate ZIKV central nervous system infections in human stem cell-derived cerebral organoids which recapitulate complex 3-dimensional neural architecture. Methods We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize cerebral organoids infected with ZIKV. Using this sequencing method, and published transcriptional profiles, we identify multiple cellular populations in our organoids, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, defining transcriptional features of uninfected cells and infected cells. Results In this model of the developing brain, we identify preferred tropisms of ZIKV infection and pronounced effects on cell division, differentiation, and death. Our data additionally reveal differences in cellular populations and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. This finding might help explain phenotypic differences attributed to the viruses, including variable propensity to cause microcephaly. Conclusion Overall, our work provides insight into normal and diseased human brain development, and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection. Disclosures All Authors: No reported Disclosures.

scholarly journals Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis

Circulation ◽  
2020 ◽  
Vol 142 (21) ◽  
pp. 2045-2059 ◽  
Author(s):  
Gabriel F. Alencar ◽  
Katherine M. Owsiany ◽  
Santosh Karnewar ◽  
Katyayani Sukhavasi ◽  
Giuseppe Mocci ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Late Stage ◽  
Lineage Tracing ◽  
Stem Cell Marker ◽  
Cell Phenotype ◽  
Phenotypic Changes ◽  
Atherosclerotic Lesions ◽  
Single Cell Rna Sequencing

Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11 - , Lgals3 + population with a chondrocyte-like gene signature that was markedly reduced with SMC- Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene–positive, extracellular matrix-remodeling, “pioneer” cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 + osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

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