Real-Time Quantitative PCR Analyis of Neonatal Rat Aortic Neurospheres (ANS)

Progenitor/stem cell populations of the aorta have been poorly characterized and their self-renewal factors unknown. In this report, we demonstrate that sphere forming progenitor/stem cells, here within termed aortic neurospheres (ANS), can be cultivated from the aorta that are similar to skin-derived precursors (SKPs). Extensive mRNA transcript profiling revealed that ANS abundantly expressed the Inhibin βA transcript, of which the secreted protein dominantly formed the Inhibin βA-βA complex, or Activin A, in the proliferation medium. Co-localization of Activin A, Nestin and Sox2 was observed in the adventitial and peri-adventitial regions of the aorta, indicative of a progenitor/stem cell niche in vivo. Blockade of Activin A signaling, through the neutralization of the Type II Activin receptors ActRIIA and ActRIIB, resulted in the asymmetric differentiation, and epithelial-to-mesenchymal transition of the ANS, respectively. When treated with the latter neutralizing antibody, translocation of the ActRIIB receptor to the nucleus was observed in cells on the periphery of the sphere. Examination of the ActRIIB protein sequence showed the presence of a putative nuclear localization signal of the PY family that was conserved across species. The results from this study demonstrate that Activin A is a self-renewal factor required for maintaining ANS progenitor/stem cell identity and multipotency. ANS cultivated from neonatal rat aortas were cultured in defined proliferation medium containing EGF, bFGF and B27. qPCR gene expression profiling. RNA was isolated from 3 separate ANS cultures.