Ontology highlight
ABSTRACT:
PROVIDER: PRJNA722271 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14244432_1.fastq.gz | Fastqsanger.gz | |||
| SRR14244432_2.fastq.gz | Fastqsanger.gz | |||
| SRR14244433_1.fastq.gz | Fastqsanger.gz | |||
| SRR14244433_2.fastq.gz | Fastqsanger.gz | |||
| SRR14244434_1.fastq.gz | Fastqsanger.gz |
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Project description:The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelium (evHCEnC) with that of primary HCEnC and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level primary HCEnC most closely resemble evHCEC and thus represent a viable therapeutic option for managing corneal endothelial dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of the potential clinical utility of the cultured HCEnC for the management of corneal endothelial cell failure. Transcriptomes from ex vivo corneal endothelium, primary cultures and three cell lines were compared. Three samples of each endothelial cell group were submitted for RNA sequencing for a total of 15 samples. The transcriptome for the ex vivo corneal endothelium was used as the reference (i.e., proxy for in vivo corneal endothelium). Transcript abundances for a subset of genes associated with corneal endothelial cell function or disease were validated with qPCR and western blot. Samples of ex vivo endothelium used for validation were independent replicates not used for RNA-sequencing.
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Project description:Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on SKPs (skin-derived precursors) a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated 5 such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKPs self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy. We obtained three independent isolates of SKPs from newborn Sprague-Dawley rat pups. Secondary SKPs spheres were dissociated, treated with 100 nM of alprostadil, trimebutine maleate or 100 nM of both trimebutine maleate and trametinib for 24 hour. RNA samples deriving from these cells were analyzed on the Affymetrix GeneChip Rat Gene 2.0 ST Array.
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Project description:Global expression analysis of neural crest-like skin-derived precursors (SKPs) and Sox2-positive follicle dermal cells that SKPs originate from. In spite of the remarkable regenerative capacity of mammalian skin, an adult dermal stem cell has not yet been identified. Here, we provide evidence that SKPs, multipotent neural crest-like skin-derived precursors, represent an adult dermal stem cell. When transplanted into adult skin, SKPs can reconstitute the adult dermis, contribute to dermal wound-healing, home to a hair follicle niche, and instruct epidermal cells to make hair follicles. Hair follicle-derived SKPs self-renew, maintain their multipotency, and serially reconstitute hair follicles. The endogenous origin of SKPs are Sox2-positive follicle dermal cells that share a similar global gene expression profile with SKPs. These endogenous cells home back to their follicle niche, induce hair follicle morphogenesis, and differentiate into neural and dermal progeny. Hair follicle-associated dermal cells will move out of their niche to contribute to dermal maintenance and wound-healing. These studies therefore identify a dermal stem cell, and provide a biological rationale for the presence of a multipotent precursor within adult dermis, findings with important therapeutic implications.
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Project description:Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally-derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally-derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from facial (neural crest-derived) and foreskin (mesodermally-derived) dermis, and the mesodermally-derived SKPs can make myelinating Schwann cells. Thus, non-neural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally-defined lineage boundaries are more flexible than widely thought.
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