A single cell transcriptome atlas reveals the heterogeneity of the healthy human cornea and identifies novel markers of the corneal limbus and stroma

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman layer, stroma, Descemet membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified that the expression of CAV1, CXCL14, HOMER3 and CPVL were exclusive to the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

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Single cell transcriptomics reveals the heterogeneity of the human cornea to identify novel markers of the limbus and stroma

Scientific Reports ◽

10.1038/s41598-021-01015-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pere Català ◽

Nathalie Groen ◽

Jasmin A. Dehnen ◽

Eduardo Soares ◽

Arianne J. H. van Velthoven ◽

...

Keyword(s):

Single Cell ◽

Cell Types ◽

Human Cornea ◽

Corneal Tissue ◽

Corneal Epithelial ◽

Disease States ◽

Heterogeneous Tissue ◽

Cell Niche ◽

Different Cell Types ◽

Regenerative Therapies

AbstractThe cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

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Single-cell transcriptome analysis of the zebrafish embryonic trunk

10.1101/2021.05.07.443129 ◽

2021 ◽

Author(s):

Sanjeeva S Metikala ◽

Satish Casie Chetty ◽

Saulius Sumanas

Keyword(s):

Single Cell ◽

Molecular Mechanisms ◽

Cell Lineage ◽

Cell Types ◽

Rna Seq ◽

Cell Lineages ◽

Distinct Cell ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Different Cell Types

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.

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Single-cell transcriptome analysis of the zebrafish embryonic trunk

PLoS ONE ◽

10.1371/journal.pone.0254024 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254024

Author(s):

Sanjeeva Metikala ◽

Satish Casie Chetty ◽

Saulius Sumanas

Keyword(s):

Single Cell ◽

Molecular Mechanisms ◽

Cell Lineage ◽

Cell Types ◽

Rna Seq ◽

Cell Lineages ◽

Distinct Cell ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Different Cell Types

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scRNASeqDB: a database for gene expression profiling in human single cell by RNA-seq

10.1101/104810 ◽

2017 ◽

Cited By ~ 7

Author(s):

Yuan Cao ◽

Junjie Zhu ◽

Guangchun Han ◽

Peilin Jia ◽

Zhongming Zhao

Keyword(s):

Single Cell ◽

Expression Profiles ◽

Cell Types ◽

Or Groups ◽

Cell Transcriptome ◽

Almost All ◽

Single Cell Transcriptome ◽

Different Cell Types ◽

Online Web

AbstractSummary: Single-cell RNA sequencing (scRNA-Seq) is quickly becoming a powerful tool for high-throughput transcriptomic analysis of cell states and dynamics. Both the number and quality of scRNA-Seq datasets have dramatically increased recently. So far, there is no database that comprehensively collects and curates scRNA-Seq data in humans. Here, we present scRNASeqDB, a database that includes almost all the currently available human single cell transcriptome datasets (n= 36) covering 71 human cell lines or types and 8910 samples. Our online web interface allows user to query and visualize expression profiles of the gene(s) of interest, search for genes that are expressed in different cell types or groups, or retrieve differentially expressed genes between cell types or groups. The scRNASeqDB is a valuable resource for single cell transcriptional studies.Availability: The database is available at https://bioinfo.uth.edu/scrnaseqdb/.Contact: [email protected]

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High-Throughput Single-Cell Transcriptome Profiling of Plant Cell Types

Cell Reports ◽

10.1016/j.celrep.2019.04.054 ◽

2019 ◽

Vol 27 (7) ◽

pp. 2241-2247.e4 ◽

Cited By ~ 57

Author(s):

Christine N. Shulse ◽

Benjamin J. Cole ◽

Doina Ciobanu ◽

Junyan Lin ◽

Yuko Yoshinaga ◽

...

Keyword(s):

Single Cell ◽

Plant Cell ◽

High Throughput ◽

Transcriptome Profiling ◽

Cell Types ◽

Cell Transcriptome ◽

Single Cell Transcriptome

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Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells’ heterogeneity

Nature Communications ◽

10.1038/s41467-020-18207-z ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Yehuda Schlesinger ◽

Oshri Yosefov-Levi ◽

Dror Kolodkin-Gal ◽

Roy Zvi Granit ◽

Luriano Peters ◽

...

Keyword(s):

Single Cell ◽

Malignant Lesion ◽

Tumor Development ◽

Initial Step ◽

Cell Types ◽

Tumor Formation ◽

Specific Cell ◽

Preinvasive Lesions ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Abstract Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.

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Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants

10.1101/2021.09.27.461256 ◽

2021 ◽

Author(s):

Zhouhuan Xi ◽

Bilge E. Ozturk ◽

Molly E. Johnson ◽

Leah C. Byrne

Keyword(s):

Single Cell ◽

Viral Vector ◽

Cell Types ◽

Adeno Associated Virus ◽

Altered Expression ◽

Gene Therapies ◽

Retinal Tissue ◽

Retinal Explants ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.

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A Single-Cell Transcriptome Atlas for Zebrafish Development

10.1101/738344 ◽

2019 ◽

Cited By ~ 1

Author(s):

Dylan R. Farnsworth ◽

Lauren Saunders ◽

Adam C. Miller

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Developmental Time ◽

Cell Type ◽

Specific Expression ◽

Transcriptional Profiles ◽

Larval Stages ◽

Cell Transcriptome ◽

Single Cell Transcriptome

ABSTRACTThe ability to define cell types and how they change during organogenesis is central to our understanding of animal development and human disease. Despite the crucial nature of this knowledge, we have yet to fully characterize all distinct cell types and the gene expression differences that generate cell types during development. To address this knowledge gap, we produced an Atlas using single-cell RNA-sequencing methods to investigate gene expression from the pharyngula to early larval stages in developing zebrafish. Our single-cell transcriptome Atlas encompasses transcriptional profiles from 44,102 cells across four days of development using duplicate experiments that confirmed high reproducibility. We annotated 220 identified clusters and highlighted several strategies for interrogating changes in gene expression associated with the development of zebrafish embryos at single-cell resolution. Furthermore, we highlight the power of this analysis to assign new cell-type or developmental stage-specific expression information to many genes, including those that are currently known only by sequence and/or that lack expression information altogether. The resulting Atlas is a resource of biologists to generate hypotheses for genetic (mutant) or functional analysis, to launch an effort to define the diversity of cell-types during zebrafish organogenesis, and to examine the transcriptional profiles that produce each cell type over developmental time.

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A single cell transcriptome atlas of the developing zebrafish hindbrain

10.1101/745141 ◽

2019 ◽

Cited By ~ 1

Author(s):

Monica Tambalo ◽

Richard Mitter ◽

David G. Wilkinson

Keyword(s):

Single Cell ◽

Neuronal Differentiation ◽

Cell Types ◽

Spatial Patterning ◽

Rna Seq ◽

Valuable Resource ◽

Dorsoventral Axis ◽

Single Cell Rna Sequencing ◽

Cell Transcriptome ◽

Single Cell Transcriptome

AbstractSegmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at bounderies and segment centres, in part mediated by Fgf20 signaling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.

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scAAVengr: Single-cell transcriptome-based quantification of engineered AAVs in non-human primate retina

10.1101/2020.10.01.323196 ◽

2020 ◽

Author(s):

Bilge E. Öztürk ◽

Molly E. Johnson ◽

Michael Kleyman ◽

Serhan Turunç ◽

Jing He ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Adeno Associated Virus ◽

Gene Therapies ◽

Naturally Occurring ◽

Cell Transcriptome ◽

Large Animals ◽

Single Cell Transcriptome ◽

Human Primate

AbstractAdeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

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