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A novel single-cell RNA-sequencing approach and its applicability connecting genotype to phenotype in ageing disease.

ABSTRACT: Single cell multi-omics analysis has the potential to yield a comprehensive understanding of the cellular events that underlie the basis of human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. Most currently used single-cell RNA-sequencing platforms have limitations in several areas including cell selection, documentation and library chemistry. In this study, we describe a novel high-throughput, full-length, single-cell RNA-sequencing approach that combines the CellenONE isolation and sorting system with the ICELL8 processing instrument. This method offers substantial improvements in single cell selection, documentation and capturing rate. Moreover, it allows the use of flexible chemistry for library preparations and the analysis of living or fixed cells, whole cells independent of sizing and morphology, as well as of nuclei. We applied this method to dermal fibroblasts derived from six patients with different segmental progeria syndromes and defined phenotype associated pathway signatures with variant associated expression modifiers. These results validate the applicability of our method to highlight genotype-expression relationships for molecular phenotyping of individual cells derived from human patients.

SUBMITTER: Shomroni O

PROVIDER: S-EPMC8904555 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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A novel single-cell RNA-sequencing approach and its applicability connecting genotype to phenotype in ageing disease.

Shomroni Orr O Sitte Maren M Schmidt Julia J Parbin Sabnam S Ludewig Fabian F Yigit Gökhan G Zelarayan Laura Cecilia LC Streckfuss-Bömeke Katrin K Wollnik Bernd B Salinas Gabriela G

Scientific reports 20220308 1

Single cell multi-omics analysis has the potential to yield a comprehensive understanding of the cellular events that underlie the basis of human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. Most currently used single-cell RNA-sequencing platforms have limitations in several areas including cell selection, documentation and library chemistry. In this study, we describe a novel high-throughput, full-length, ...[more]

PMID: 35260714

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Project description:Study questionWhat is the molecular landscape underlying the functional decline of human testicular ageing?Summary answerThe present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing.What is known alreadyTesticular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing.Study design, size, durationHuman testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors.Participants/materials, setting, methodsscRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat.Main results and the role of chanceThe single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing.Large scale dataThe scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349.Limitations, reasons for cautionOwing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future.Wider implications of the findingsThese findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism.Study funding/competing interest(s)This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest.

Project description:BACKGROUND: Lymnaeid snails transmit medical and veterinary important trematodiases, mainly fascioliasis. Vector specificity of fasciolid parasites defines disease distribution and characteristics. Different lymnaeid species appear linked to different transmission and epidemiological patterns. Pronounced susceptibility differences to absolute resistance have been described among lymnaeid populations. When assessing disease characteristics in different endemic areas, unexpected results were obtained in studies on lymnaeid susceptibility to Fasciola. We undertook studies to understand this disease transmission heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A ten-year study in Iran, Egypt, Spain, the Dominican Republic, Mexico, Venezuela, Ecuador and Peru, demonstrated that such heterogeneity is not due to susceptibility differences, but to a hitherto overlooked cryptic species, Lymnaea schirazensis, confused with the main vector Galba truncatula and/or other Galba/Fossaria vectors. Nuclear rDNA and mtDNA sequences and phylogenetic reconstruction highlighted an old evolutionary divergence from other Galba/Fossaria species, and a low intraspecific variability suggesting a recent spread from one geographical source. Morphometry, anatomy and egg cluster analyses allowed for phenotypic differentiation. Selfing, egg laying, and habitat characteristics indicated a migration capacity by passive transport. Studies showed that it is not a vector species (n?=?8572 field collected, 20 populations): snail finding and penetration by F. hepatica miracidium occur but never lead to cercarial production (n?=?338 experimentally infected). CONCLUSIONS/SIGNIFICANCE: This species has been distorting fasciolid specificity/susceptibility and fascioliasis geographical distribution data. Hence, a large body of literature on G. truncatula should be revised. Its existence has henceforth to be considered in research. Genetic data on livestock, archeology and history along the 10,000-year post-domestication period explain its wide spread from the Neolithic Fertile Crescent. It is an efficient biomarker for the follow-up of livestock movements, a crucial aspect in fascioliasis emergence. It offers an outstanding laboratory model for genetic studies on susceptibility/resistance in F. hepatica/lymnaeid interaction, a field of applied research with disease control perspectives.

Dataset Information

A novel single-cell RNA-sequencing approach and its applicability connecting genotype to phenotype in ageing disease.

Publications

A novel single-cell RNA-sequencing approach and its applicability connecting genotype to phenotype in ageing disease.

Similar Datasets

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