SOX2+ sustentacular cells are stem cells in the postnatal adrenal medulla

Renewal of the catecholamine-secreting chromaffin cell population of the adrenal medulla is necessary for physiological homeostasis throughout life. Definitive evidence for the presence or absence of an adrenomedullary stem cell has been enigmatic. In this work, we show that a subpopulation of uncommitted sustentacular cells, previously thought to be support cells, are in fact adrenomedullary stem cells. These postnatal cells express the transcription factor SOX2, and give rise to chromaffin cells of both the adrenaline and noradrenaline lineages in vivo and in vitro. We present comprehensive transcriptomic data of the mouse adrenal medulla as well as of purified SOX2+ adrenomedullary stem cell populations. Through genetic tracing, we show that postnatal stem cells specialise from embryonic Schwann Cell Precursors and identify this population in the normal adult human adrenal medulla. Overall design: 10 adrenals from 5 mixed sex P15 mice were dissected on ice, surrounding fat and excess adrenal cortex were removed manually. Medullae were placed in an enzymatic digestion mix containing 50µg/ml DNAse I (Sigma, D5025), 10mg/ml Collagenase II (Worthington, LS004177), 2.5µg/ml Fungizone (Gibco, 15290026), 0.1X Trypsin-EDTA (Sigma, 59418C) in 1X Hank's Balanced Salt Solution (HBSS) (Gibco, 14025050) and incubated at 37°C for 15 minutes. Enzymes were inactivated by addition of 10 times volume of serum-containing Base Media: DMEM/F-12 (Gibco, 31330-038) + 5% FBS (Merk, F0804) + 50U/ml Penicillin-Streptomycin (Gibco, 15070063). The cell suspension was centrifuged at 281g for 5 minutes at room temperature, washed twice in PBS and pellets were resuspended in a solution of HBSS 2.5% FBS. An aliquot of 10,000 viable cells were used for the experiment. Library preparation and sequencing were performed by the BRC Genomics Core at KCL. Library preparation was done using the Chromium 10X Single Cell 3' Reagent Kit v3 (10x Genomics) and a Chromium Controller (10x Genomics) following the manufacturer's protocol. Once obtained, barcoded transcripts from single cells were sequenced with an Illumina HiSeq 2500.