Speed Poster ESA-SRB-APEG-NZSE 2022

Mapping the single cell transcriptome of murine adrenal glands (#80)

Lara E Graves 1 2 3 , Nader Aryamanesh 4 5 , Shubha Srinivasan 2 3 , Samantha Ginn 1 5 , Ian E Alexander 1 2 6
  1. Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, Westmead, NSW, Australia
  2. Children’s Hospital Westmead Clinical School, University of Sydney, Westmead, New South Wales, Australia
  3. Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Westmead, NEW SOUTH WALES, Australia
  4. Embryology Unit, Bioinformatics Support Group, Children’s Medical Research Institute, Westmead, NSW, Australia
  5. School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
  6. Discipline of Genetic Medicine, Children’s Hospital Westmead Clinical School, University of Sydney, Westmead, NSW, Australia

The adrenal cortex undergoes constant renewal(1): capsular stem cells expressing GL1+ give rise to cortical SF1+ progenitor and SF1+ differentiated steroidogenic cells of the zona glomerulosa (zG)(2-4). Despite knowledge of their existence, these populations are yet to be adequately described. Identification is required for the development of durable gene therapy for monogenic disorders of the adrenal cortex. As the progenitor population is very small, they do not form a distinct cell cluster in single cell RNA sequencing (scRNA-Seq) transcriptomic analysis and have not been identified by a previous group(5). The aim of this study was to map the adrenal single cell transcriptome and to identify adrenocortical progenitor cells.

Adrenal glands were harvested from wild-type mice, snap frozen, and dissociated into single nuclei. The scRNA-Seq library was prepared using 10x Chromium technology and sequenced by the Illumina NovaSeq platform. The transcriptome was mapped to the mouse mm10 reference genome using the CellRanger pipeline(6), followed by scRNA-Seq analysis using the Seurat v4.0 package. Cell types were annotated using a combination of the Clustifyr package and manual annotation using gene expression patterns.

The scRNA-Seq clustering pattern is shown in Figure 1. The progenitor population did not form a distinct cluster. However, cells co-expressing Nr5a1 (encodes SF1 protein), Shh and Ctnnb1 that did not express Cyp11b1 or Cyp11b2 (markers of differentiated adrenocortical cells) were detected in cluster 0, consistent with SF1+/SHH+ progenitors. Progenitor gene expression was detected in 0.34% and 0.22% of cells from the female and male, respectively.

In conclusion, a population consistent with adrenocortical progenitor cells was identified. Transcriptomic analysis of adrenals from 21-hydroxylase deficient mice is underway, and the resultant profile will be compared with that of the wild-type mice. Additional ACTH stimulation is postulated to affect these populations and may expand the progenitor pool.

 

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