scholarly journals Cell types of the human retina and its organoids at single-cell resolution: developmental convergence, transcriptomic identity, and disease map

2019 ◽  
Author(s):  
Cameron S. Cowan ◽  
Magdalena Renner ◽  
Brigitte Gross-Scherf ◽  
David Goldblum ◽  
Martin Munz ◽  
...  
Keyword(s):  
Single Cells ◽  
Pigment Epithelium ◽  
Cell Types ◽  
Retinal Cell ◽  
Human Retina ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Retina Development ◽  
Adult Human

SummaryHow closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the RNA of 158,844 single cells from these organoids at six developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable ‘developed’ state at a rate similar to human retina development in vivo and the transcriptomes of organoid cell types converged towards the transcriptomes of adult peripheral retinal cell types. The expression of disease-associated genes was significantly cell-type specific in adult retina and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in adult human retinas.

113. DEVELOPMENT OF A METHOD OF SEMINIFEROUS TUBULE TRANSFECTION IN VITRO TO DEFINE POSTMEIOTIC GENE REGULATION

2009 ◽  
Vol 21 (9) ◽  
pp. 32
Author(s):  
S. Danner ◽  
C. Kirchhoff ◽  
R. Ivell
Keyword(s):  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Cell Types ◽  
Seminiferous Tubules ◽  
Gene Promoters ◽  
Cell Type ◽  
Cell Type Specificity ◽  
High Throughput Analysis ◽  
Apparent Lack

Postmeiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. Here we report the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.

scholarly journals Retinal organoids: a window into human retinal development

Development ◽  
2020 ◽  
Vol 147 (24) ◽  
pp. dev189746
Author(s):  
Michelle O'Hara-Wright ◽  
Anai Gonzalez-Cordero
Keyword(s):  
Cell Fate ◽  
Developmental Trajectories ◽  
Retinal Development ◽  
Retinal Disease ◽  
Cell Types ◽  
Model Organisms ◽  
Retinal Cell ◽  
Human Retina ◽  
Cell Fate Decisions

ABSTRACTRetinal development and maturation are orchestrated by a series of interacting signalling networks that drive the morphogenetic transformation of the anterior developing brain. Studies in model organisms continue to elucidate these complex series of events. However, the human retina shows many differences from that of other organisms and the investigation of human eye development now benefits from stem cell-derived organoids. Retinal differentiation methods have progressed from simple 2D adherent cultures to self-organising micro-physiological systems. As models of development, these have collectively offered new insights into the previously unexplored early development of the human retina and informed our knowledge of the key cell fate decisions that govern the specification of light-sensitive photoreceptors. Although the developmental trajectories of other retinal cell types remain more elusive, the collation of omics datasets, combined with advanced culture methodology, will enable modelling of the intricate process of human retinogenesis and retinal disease in vitro.

scholarly journals PESCA: A scalable platform for the development of cell-type-specific viral drivers

2019 ◽  
Author(s):  
Sinisa Hrvatin ◽  
Christopher P. Tzeng ◽  
M. Aurel Nagy ◽  
Hume Stroud ◽  
Charalampia Koutsioumpa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Single Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Functional Evaluation ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Enhancer Activity ◽  
Cell Type Specific

AbstractEnhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.One sentence summaryHighly paralleled functional evaluation of enhancer activity in single cells generates new cell-type-specific tools with broad medical and scientific applications.

scholarly journals Adult Human Multipotent Neural Cells Could Be Distinguished from Other Cell Types by Proangiogenic Paracrine Effects via MCP-1 and GRO

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Sung Soo Kim ◽  
Hee-Jang Pyeon ◽  
Yoon Kyung Bae ◽  
Hyun Nam ◽  
Chung Kwon Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Types ◽  
Neural Cells ◽  
Cell Type ◽  
Paracrine Factors ◽  
Adult Human ◽  
Cell Type Specific ◽  
Functional Features

Adult human multipotent neural cells (ahMNCs) are unique cells derived from adult human temporal lobes. They show multipotent differentiation potentials into neurons and astrocytes. In addition, they possess proangiogenic capacities. The objective of this study was to characterize ahMNCs in terms of expression of cell type-specific markers, in vitro differentiation potentials, and paracrine factors compared with several other cell types including fetal neural stem cells (fNSCs) to provide detailed molecular and functional features of ahMNCs. Interestingly, the expression of cell type-specific markers of ahMNCs could not be differentiated from those of pericytes, mesenchymal stem cells (MSCs), or fNSCs. In contrast, differentiation potentials of ahMNCs and fNSCs into neural cells were higher than those of other cell types. Compared with MSCs, ahMNCs showed lower differentiation capacities into osteogenic and adipogenic cells. Moreover, ahMNCs uniquely expressed higher levels of MCP-1 and GRO family paracrine factors than fNSCs and MSCs. These high levels of MCP-1 and GRO family mediated in vivo proangiogenic effects of ahMNCs. These results indicate that ahMNCs have their own distinct characteristics that could distinguish ahMNCs from other cell types. Characteristics of ahMNCs could be utilized further in the preclinical and clinical development of ahMNCs for regenerative medicine. They could also be used as experimental references for other cell types including fNSCs.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Specificity of gene expression in adipocytes

1985 ◽  
Vol 5 (2) ◽  
pp. 419-421
Author(s):  
K M Zezulak ◽  
H Green
Keyword(s):  
Gene Expression ◽  
Cell Types ◽  
Cell Type ◽  
3T3 Cells ◽  
Cell Type Specificity ◽  
Number Of Genes ◽  
Enhanced Expression ◽  
Adipose Cells ◽  
Distinctive Phenotype

During the differentiation of preadipose 3T3 cells into adipose cells, the mRNAs for three proteins increase strikingly in abundance. To determine the degree of cell-type specificity in the expression of these mRNAs, we estimated their abundances in several nonadipose tissues of the mouse. None of these mRNAs was strictly confined to adipocytes, but the ensemble of three mRNAs was rather specific to adipocytes. Insofar as is revealed by these three markers, the distinctive phenotype of adipocytes is the result of the enhanced expression of a number of genes, none of which is completely silent in all other cell types.

E26 leukemia virus converts primitive erythroid cells into cycling multilineage progenitors

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1103-1110 ◽  
Author(s):  
Kelly M. McNagny ◽  
Thomas Graf
Keyword(s):  
Leukemia Virus ◽  
Yolk Sac ◽  
Erythroid Cells ◽  
Multilineage Differentiation ◽  
Cell Type ◽  
Hematopoietic Progenitors ◽  
Cell Type Specificity ◽  
Erythroid Progenitors ◽  
Avian Erythroblastosis Virus

Abstract Acute chicken leukemia retroviruses, because of their capacity to readily transform hematopoietic cells in vitro, are ideal models to study the mechanisms governing the cell-type specificity of oncoproteins. Here we analyzed the transformation specificity of 2 acute chicken leukemia retroviruses, the Myb-Ets– encoding E26 virus and the ErbA/ErbB-encoding avian erythroblastosis virus (AEV). While cells transformed by E26 are multipotent (designated “MEP” cells), those transformed by AEV resemble erythroblasts. Using antibodies to separate subpopulations of precirculation yolk sac cells, both viruses were found to induce the proliferation of primitive erythroid progenitors within 2 days of infection. However, while AEV induced a block in differentiation of the cells, E26 induced a gradual shift in their phenotype and the acquisition of the potential for multilineage differentiation. These results suggest that the Myb-Ets oncoprotein of the E26 leukemia virus converts primitive erythroid cells into proliferating definitive-type multipotent hematopoietic progenitors.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

scholarly journals A scalable platform for the development of cell-type-specific viral drivers

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sinisa Hrvatin ◽  
Christopher P Tzeng ◽  
M Aurel Nagy ◽  
Hume Stroud ◽  
Charalampia Koutsioumpa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
High Specificity ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Cell Type Specific ◽  
The Many ◽  
Dna Regulatory Elements

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

scholarly journals Genomic Architecture of Cells in Tissues (GeACT): Study of Human Mid-gestation Fetus

2020 ◽  
Author(s):  
Feng Tian ◽  
Fan Zhou ◽  
Xiang Li ◽  
Wenping Ma ◽  
Honggui Wu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Human Cell ◽  
Expression Profiles ◽  
Single Cells ◽  
Cell Types ◽  
List Type ◽  
Cell Type ◽  
Genomic Architecture ◽  
Gene Modules

SummaryBy circumventing cellular heterogeneity, single cell omics have now been widely utilized for cell typing in human tissues, culminating with the undertaking of human cell atlas aimed at characterizing all human cell types. However, more important are the probing of gene regulatory networks, underlying chromatin architecture and critical transcription factors for each cell type. Here we report the Genomic Architecture of Cells in Tissues (GeACT), a comprehensive genomic data base that collectively address the above needs with the goal of understanding the functional genome in action. GeACT was made possible by our novel single-cell RNA-seq (MALBAC-DT) and ATAC-seq (METATAC) methods of high detectability and precision. We exemplified GeACT by first studying representative organs in human mid-gestation fetus. In particular, correlated gene modules (CGMs) are observed and found to be cell-type-dependent. We linked gene expression profiles to the underlying chromatin states, and found the key transcription factors for representative CGMs.HighlightsGenomic Architecture of Cells in Tissues (GeACT) data for human mid-gestation fetusDetermining correlated gene modules (CGMs) in different cell types by MALBAC-DTMeasuring chromatin open regions in single cells with high detectability by METATACIntegrating transcriptomics and chromatin accessibility to reveal key TFs for a CGM

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