Expression of progenitor markers is associated with the functionality of a bioartificial adrenal cortex

Abstract

Encapsulation of primary bovine adrenocortical cells in alginate is an efficacious model of a bioartificial adrenal cortex. Such a bioartificial adrenal cortex can be used for the restoration of lost adrenal function in vivo as well as for in vitro modeling of the adrenal microenvironment and for investigation of cell-cell interactions in the adrenals. The aim of this work was the optimization of a bioartificial adrenal cortex, that is the generation of a highly productive, self-regenerating, long-term functioning and immune tolerant bioartificial organ. To achieve this, it is necessary that adrenocortical stem and progenitor cells are present in the bioartificial gland, as these undifferentiated cells play important roles in the function of the mature gland. Here, we verified the presence of adrenocortical progenitors in cultures of bovine adrenocortical cells, studied the dynamics of their appearance and growth and determined the optimal time point for cell encapsulation. These procedures increased the functional life span and reduced the immunogenicity of the bioartificial adrenal cortex. This model allows the use of the luteinizing hormone-releasing hormone (LHRH) agonist triptorelin, the neuropeptide bombesin, and retinoic acid to alter cell number and the release of cortisol over long periods of time.

Fig 1. Dynamics of the relative gene expression of progenitor markers in primary cultures of bovine adrenocortical cells.

RT-PCR analysis of the expression of Patched1 (A), GLI1 (B), nestin (C) and DAX1 (D) in in BAC cultures for a period of 10 days after cell isolation. All data presented as mean ± SEM, n≥3 for each sample. Reference gene RPS9.

Fig 2. Dynamics of the relative mRNA gene expression of SF1, CYP17A1 and interleukins 6 and 1β during cultivation of BAC.

A+B–Dynamics of SF1 (A) and CYP17A1 (B) expression with and without ACTH-stimulation for a period of 10 days after cell isolation. C–Downregulation of progenitor markers after ACTH-stimulation on day 7 after cell isolation. D–Dynamics of the gene expression of IL-6 and IL-1β 10 days after cell isolation. All data presented as mean ± SEM, n≥6 for each sample, *p≤0.05, ***p≤0.001. Reference gene RPS9.

Fig 3. Comparative analysis of bioartificial adrenal cortices, created from BAC cultures, cultivated for 1 or 7 days after isolation before encapsulation in alginate.

A–Dynamics of ACTH-stimulated cortisol production of bioartificial adrenal cortices during a cultivation period of 114 days (All data presented as mean ± SEM; n≥5 for each time point; ***p≤0.001). B—Dynamics of the basal cortisol release of bioartificial adrenal cortices during a cultivation period of 114 days (n≥5 for each time point). C–Cell cluster formation in bioartificial adrenal cortices formed from BACs 1 or 7 days after cell isolation (FDA/PI staining plus light microscopy). Scale bars = 100 μm. Uncropped images are seen in S4 Fig. D–Positive CYP11B1 staining (green) of cell clusters (Immunofluorescence plus light microscopy). Scale bar = 20 μm.

Fig 4. Short- and long-term effects of triptorelin, bombesin and retinoic acid on bioartificial adrenal cortices.

A–Short-term effect of the compounds on both basal and ACTH-stimulated cortisol release within the first two weeks of treatment (all data presented as mean ± SEM, n≥18 for each group, *p≤0.05, **p≤0.01). B–Long-term effect of the compounds on basal and ACTH-stimulated cortisol levels at the end point of the experiment (112–114 days of cultivation). All data presented as mean ± SEM, n = 5 for each group, *p≤0.05. C–Cell cluster formation in different groups of bioartificial adrenal cortices after cultivation for three months (FDA/PI staining). Scale bars = 100 μm. Uncropped images are seen in S4 Fig. D—Amount of cells, counted in bioartificial adrenal cortices after cultivation for 114 days (n = 6 for each group).