Endocrine Abstracts (2007) 14 P112

Isolation of the Side population (SP) from murine adrenal glands renders cells with adrenocortical stem cell properties

Urs Lichtenauer1, Igor Shapiro1, Klaus Geiger2, Anne-Marie Pulichino3, Jaques Drouin3 & Felix Beuschlein1

1Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany, 2Department of Internal Medicine I, Albert-Ludwigs-University Freiburg, Freiburg, Germany, 3Laboratoire de Genetique Moleculaire, Institut de Recherches Cliniques de Montreal, Montral, Canada.

Radioactive and transgenic tracing experiments indicate that in the adult adrenal stem cells persist in the periphery of the cortex, which migrate centripetally and populate the inner cortical zones upon differentiation. However, investigation of these cells has been hampered by the lack of known marker genes. Vital Hoechst dye exclusion has been described as a method for isolating a side population (SP) from mouse bone marrow, which was enriched with stem cells. Utilizing this technique, we demonstrate the presence of SP cells in a variety of adrenal derived cell populations including normal mouse (0.71–0.83%) and human (0.01%) adrenals. After FACS sorting, isolation of SP and non SP (NSP) cells from murine adrenal glands revealed self-renewal and long-term culture capacities only for the SP fraction, which grew in a fibroblast-like manner, whereas the NSP cells did not proliferate. In addition, adrenal SP cells expressed adrenocortical markers such as MC2 receptor, StAR, and P450scc by means of RT-PCR and IHC. Interestingly, in a mouse model of ACTH deficiency (Tpit knock out animals, Tpit−/−), the proportion of SP cells was significant higher in comparison to heterozygous animals (Tpit−/− 0.45±0.16% vs. Tpit+/− 0.13±0.04%;P<0.004). This higher SP cell proportion was associated with an increased width of the subcapsular cell compartment (Tpit−/− 100±12.3% vs. Tpit+/− 259±10.7%; P<0.0001), which was characterized by the lack of expression of steroidogenic enzymes such as 3ßHSD. Short term ACTH treatment of Tpit−/− animals resulted in a decrease of SP proportion (0.09%) and a shrinkage of the sub-capsular zone similar to that of untreated Tpit+/− controls (130±10.2%;P=0.33). In summary, the adrenal Side Population displays certain stem cell properties. Moreover, we present indirect evidence that ACTH might be required for adrenocortical stem cell differentiation thus affecting adrenal zonation in vivo. Current studies including in vitro stimulation and in vivo transplantation experiments aim at the further characterization of this cell population.

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