Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium—retinal organoid—that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

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Single-nuclei RNA-seq on human retinal tissue provides improved transcriptome profiling

Nature Communications ◽

10.1038/s41467-019-12917-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Qingnan Liang ◽

Rachayata Dharmat ◽

Leah Owen ◽

Akbar Shakoor ◽

Yumei Li ◽

...

Keyword(s):

Single Cell ◽

Transcriptome Profiling ◽

Cell Types ◽

Retinal Cell ◽

Peripheral Retina ◽

Marker Genes ◽

Rna Seq ◽

Cell Type ◽

Retinal Tissue ◽

The Individual

AbstractSingle-cell RNA-seq is a powerful tool in decoding the heterogeneity in complex tissues by generating transcriptomic profiles of the individual cell. Here, we report a single-nuclei RNA-seq (snRNA-seq) transcriptomic study on human retinal tissue, which is composed of multiple cell types with distinct functions. Six samples from three healthy donors are profiled and high-quality RNA-seq data is obtained for 5873 single nuclei. All major retinal cell types are observed and marker genes for each cell type are identified. The gene expression of the macular and peripheral retina is compared to each other at cell-type level. Furthermore, our dataset shows an improved power for prioritizing genes associated with human retinal diseases compared to both mouse single-cell RNA-seq and human bulk RNA-seq results. In conclusion, we demonstrate that obtaining single cell transcriptomes from human frozen tissues can provide insight missed by either human bulk RNA-seq or animal models.

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Deciphering cell lineage specification during male sex determination with single-cell RNA sequencing

10.1101/190264 ◽

2017 ◽

Cited By ~ 2

Author(s):

Isabelle Stévant ◽

Yasmine Neirjinck ◽

Christelle Borel ◽

Jessica Escoffier ◽

Lee B. Smith ◽

...

Keyword(s):

Sex Determination ◽

Single Cell ◽

Progenitor Cells ◽

Rna Sequencing ◽

Cell Fate ◽

Time Course ◽

Cell Lineage ◽

Lineage Specification ◽

Cell Fate Decisions ◽

Single Cell Rna Sequencing

SummaryThe gonad is a unique biological system for studying cell fate decisions. However, major questions remain regarding the identity of somatic progenitor cells and the transcriptional events driving cell differentiation. Using time course single cell RNA sequencing on XY mouse gonads during sex determination, we identified a single population of somatic progenitor cells prior sex determination. A subset of these progenitors differentiate into Sertoli cells, a process characterized by a highly dynamic genetic program consisting of sequential waves of gene expression. Another subset of multipotent cells maintains their progenitor state but undergo significant transcriptional changes that restrict their competence towards a steroidogenic fate required for the differentiation of fetal Leydig cells. These results question the dogma of the existence of two distinct somatic cell lineages at the onset of sex determination and propose a new model of lineage specification from a unique progenitor cell population.

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Faculty Opinions recommendation of Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733883362.793555704 ◽

2019 ◽

Author(s):

Miles Wilkinson

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing ◽

Human Spermatogenesis

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Single-Cell RNA Sequencing in Human Retinal Degeneration Reveals Distinct Glial Cell Populations

Cells ◽

10.3390/cells9020438 ◽

2020 ◽

Vol 9 (2) ◽

pp. 438 ◽

Cited By ~ 3

Author(s):

Andrew P. Voigt ◽

Elaine Binkley ◽

Miles J. Flamme-Wiese ◽

Shemin Zeng ◽

Adam P. DeLuca ◽

...

Keyword(s):

Single Cell ◽

Retinal Degeneration ◽

Rna Sequencing ◽

Glial Cell ◽

Glial Cells ◽

Peripheral Retina ◽

Cell Populations ◽

Specific Gene ◽

Autoimmune Retinopathy ◽

Single Cell Rna Sequencing

Degenerative diseases affecting retinal photoreceptor cells have numerous etiologies and clinical presentations. We clinically and molecularly studied the retina of a 70-year-old patient with retinal degeneration attributed to autoimmune retinopathy. The patient was followed for 19 years for progressive peripheral visual field loss and pigmentary changes. Single-cell RNA sequencing was performed on foveal and peripheral retina from this patient and four control patients, and cell-specific gene expression differences were identified between healthy and degenerating retina. Distinct populations of glial cells, including astrocytes and Müller cells, were identified in the tissue from the retinal degeneration patient. The glial cell populations demonstrated an expression profile consistent with reactive gliosis. This report provides evidence that glial cells have a distinct transcriptome in the setting of human retinal degeneration and represents a complementary clinical and molecular investigation of a case of progressive retinal disease.

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Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for Caenorhabditis elegans neuron classes

Nucleic Acids Research ◽

10.1093/nar/gkaa486 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ramiro Lorenzo ◽

Michiho Onizuka ◽

Matthieu Defrance ◽

Patrick Laurent

Keyword(s):

Caenorhabditis Elegans ◽

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Single Neuron ◽

Genetic Manipulations ◽

C Elegans ◽

Single Cell Rna Sequencing ◽

Normal Differentiation

Abstract Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data and on a molecular atlas describing the expression pattern of ∼800 genes at the single cell resolution, we designed an iterative clustering analysis aiming to match each cell-cluster to the ∼100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 97 of the 118 neuron classes to a cluster. Sixty two clusters were assigned to a single neuron class and 15 clusters grouped neuron classes sharing close molecular signatures. Pseudotime analysis revealed a maturation process occurring in some neurons (e.g. PDA) during the L2 stage. Based on the molecular profiles of all identified neurons, we predicted cell fate regulators and experimentally validated unc-86 for the normal differentiation of RMG neurons. Furthermore, we observed that different classes of genes functionally diversify sensory neurons, interneurons and motorneurons. Finally, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations.

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Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

Nature Communications ◽

10.1038/s41467-020-20448-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dandan Liang ◽

Jinfeng Xue ◽

Li Geng ◽

Liping Zhou ◽

Bo Lv ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Sinoatrial Node ◽

Induced Pluripotent Stem Cell ◽

Transcriptome Profiling ◽

Core Gene ◽

Cell Cluster ◽

Critical Function ◽

Gene Regulation Network ◽

Single Cell Rna Sequencing

AbstractBioelectrical impulses intrinsically generated within the sinoatrial node (SAN) trigger the contraction of the heart in mammals. Though discovered over a century ago, the molecular and cellular features of the SAN that underpin its critical function in the heart are uncharted territory. Here, we identify four distinct transcriptional clusters by single-cell RNA sequencing in the mouse SAN. Functional analysis of differentially expressed genes identifies a core cell cluster enriched in the electrogenic genes. The similar cellular features are also observed in the SAN from both rabbit and cynomolgus monkey. Notably, Vsnl1, a core cell cluster marker in mouse, is abundantly expressed in SAN, but is barely detectable in atrium or ventricle, suggesting that Vsnl1 is a potential SAN marker. Importantly, deficiency of Vsnl1 not only reduces the beating rate of human induced pluripotent stem cell - derived cardiomyocytes (hiPSC-CMs) but also the heart rate of mice. Furthermore, weighted gene co-expression network analysis (WGCNA) unveiled the core gene regulation network governing the function of the SAN in mice. Overall, these findings reveal the whole transcriptome profiling of the SAN at single-cell resolution, representing an advance toward understanding of both the biology and the pathology of SAN.

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Epigenetic adaptation prolongs photoreceptor survival during retinal degeneration

10.1101/774950 ◽

2019 ◽

Author(s):

Rachayata Dharmat ◽

Sangbae Kim ◽

Hehe Liu ◽

Shangyi Fu ◽

Yumei Li ◽

...

Keyword(s):

Single Cell ◽

Exponential Distribution ◽

Time Course ◽

Chromatin State ◽

Retinal Cell ◽

Marker Genes ◽

Specific Marker ◽

Stretched Exponential ◽

Rod Cells ◽

Stretched Exponential Distribution

AbstractNeural degenerative diseases often display a progressive loss of cells as a stretched exponential distribution. The mechanisms underlying the survival of a subset of genetically identical cells in a population beyond what is expected by chance alone remains unknown. To gain mechanistic insights underlying prolonged cellular survival, we used Spata7 mutant mice as a model and performed single-cell transcriptomic profiling of retinal tissue along the time course of photoreceptor degeneration. Intriguingly, rod cells that survive beyond the initial rapid cell apoptosis phase progressively acquire a distinct transcriptome profile. In these rod cells, expression of photoreceptor-specific phototransduction pathway genes is downregulated while expression of other retinal cell type-specific marker genes is upregulated. These transcriptomic changes are achieved by modulation of the epigenome and changes of the chromatin state at these loci, as indicated by immunofluorescence staining and single-cell ATAC-seq. Consistent with this model, when induction of the repressive epigenetic state is blocked by in vivo histone deacetylase inhibition, all photoreceptors in the mutant retina undergo rapid degeneration, strongly curtailing the stretched exponential distribution. Our study reveals an intrinsic mechanism by which neural cells progressively adapt to genetic stress to achieve prolonged survival through epigenomic regulation and chromatin state modulation.

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Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression

10.1101/630996 ◽

2019 ◽

Cited By ~ 10

Author(s):

Anna SE Cuomo ◽

Daniel D Seaton ◽

Davis J McCarthy ◽

Iker Martinez ◽

Marc Jan Bonder ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Cell Biology ◽

Population Variation ◽

Ips Cell ◽

Cell Fate Decisions ◽

Common Genetic Variants ◽

Single Cell Rna Sequencing

AbstractRecent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts.

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Regulation of neurogenesis and gliogenesis by the matricellular protein CCN2 in the mouse retina

10.1101/2021.04.01.438112 ◽

2021 ◽

Author(s):

Golam Mohiuddin ◽

Genesis Lopez ◽

Jose Sinon ◽

M. Elizabeth Hartnett ◽

Anastasiia Bulakhova ◽

...

Keyword(s):

Glial Cell ◽

Cell Fate ◽

Matrix Protein ◽

Ex Vivo ◽

Mouse Genetics ◽

Extracellular Matrix Protein ◽

Retinal Cell ◽

Mouse Retina ◽

Matricellular Protein ◽

Retinal Progenitor

AbstractCellular communication network (CCN) 2 is an extracellular matrix protein with cell type- and context-dependent functions. Using a combination of mouse genetics and omic approaches, we show that CCN2 is expressed in early embryonic retinal progenitor cells (RPCs) and becomes restricted to fully differentiated Müller glial cells (MGCs) thereafter. Germline deletion of CCN2 in mice decreases BrdU labeling, reduces RPC pool, and impairs the competency of remaining RPCs to generate early and late born retinal cell types. Retinal hypocellularity and microphthalmia ensue. The transcriptomic changes associated with CCN2 inactivation include reduced marker and transcriptional regulator genes of retinal ganglion cells, photoreceptors and MGCs. Yap (Yes-associated protein), a singular node for transcriptional regulation of growth and differentiation genes, is also a target of CCN2 signals. In an organotypic model of ex vivo cultured embryonic retinas, CCN2 and YAP immunoreactivity signals overlap. Lentivirus-mediated YAP expression in CCN2-deficient retinal explants increases the number of differentiating Sox9-positive MGCs. Taken together, our data indicate that CCN2 controls the proliferative and differentiation potentials of RPCs ultimately endowing, a subpopulation thereof, with Müller glial cell fate.Summary statementA CCN2-YAP regulatory axis controls retinal progenitor cell growth and lineage commitment to neuronal and glial cell fates.

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