Introduction: Steroid hormones synthesized and secreted by the adrenal cortex play indispensable roles in maintaining physiological homeostasis, including the regulation of glucose metabolism, immune function and stress responses. Histologically, the adrenal cortex is characterized by a rich vascular endothelial network, with steroidogenic cells located in close proximity to endothelial cells. The transcription of all steroidogenic enzymes is regulated by Nuclear receptor 5A1 (NR5A1, also known as Ad4BP/SF1), a master regulator critical for the development of adrenal glands and gonads. Primary adrenal insufficiency, which causes steroid hormone deficiency, is currently treated with lifelong hormone replacement therapy. While this is an established medical intervention, it is associated with metabolic side effects. Moreover, long-term administration suppresses the hypothalamic-pituitary-adrenal axis and fails to accurately replicate physiological hormone levels. Insufficient replacement may lead to adrenal crisis, which can be life-threatening. Therefore, cell therapy using transplanted steroidogenic cells responsive to endogenous ACTH and other physiological stimuli is a promising alternative for hormone replacement.

Method: Murine and human mesenchymal stromal cells (MSCs) were transduced with NR5A1 via viral vectors to induce steroidogenic potential. Steroid hormone production and expression levels of key steroidogenic enzymes were assessed. Expression of the melanocortin 2 receptor (MC2R), which mediates ACTH responsiveness, was evaluated alongside in vitro ACTH-stimulated steroidogenesis. To evaluate therapeutic potential in vivo, spheres of NR5A1-induced steroidogenic cells were implanted beneath the renal capsule in bilateral adrenalectomized murine models. Post-implantation survival and circulating steroid hormone concentrations were measured. Additionally, co-culture experiments with vascular endothelial cells were performed to assess their influence on glucocorticoid production.

Results: Ectopic expression of NR5A1 in MSCs induced production of both adrenal and gonadal steroids and upregulated the expression levels of steroidogenic enzymes. In vitro, ACTH stimulation enhanced steroidogenesis. Bilateral adrenalectomized mice typically died within a few days due to adrenal insufficiency. However, implantation of NR5A1-induced steroidogenic cell spheres prolonged survival and led to detectable circulating glucocorticoid levels. Co-culture of NR5A1-induced steroidogenic cells with endothelial cells enhanced glucocorticoid, but not gonadal steroid, production. These results suggest that co-culture with endothelial cells may facilitate lineage commitment toward adrenocortical-like phenotypes.

Conclusion: Implantation of NR5A1-induced steroidogenic cells derived from mesenchymal stromal cells prolonged survival in mouse models of adrenal insufficiency, demonstrating the potential of steroid replacement via cell therapy.