ULTRASTRUCTURAL CHANGES OF THE STEM CELLS IN THE CYCLE MONOLAYER--SPHERES--MONOLAYER]

Sphere formation can be used to prepare stem cells (SCs) prior to transplantation. Here SCs isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer-spheres-monolayer cycle by transmission electron microscopy. The results obtained with both adherent-induced and hanging-drop induced spheres were similar. At first 2-3 passages (stage 1), isolated SCs displayed embryonal cell-like ultrastructure. With increasing passage times (stage 2), SCs became bigger and more electron-dark with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), prominent Golgi apparatus and numerous vacuoles. After 2 h from the initiation of the formation of spheres (stage 3), SCs gathered into clusters and formed desmosome-like intercellular contacts. Their nucleus possessed a large loose fibrillo-granular nucleoli, the cytoplasm was densely packed with disintegrated cisternae of RER, Golgi apparatus was not detected. After 24 h from the initiation of spheres (stage 4), SCs in well-formed spheres exhibited large dense nucleoli and poorly developed Golgi apparatus and RER. One day after sphere dissociating (stage 5), SCs were embryonal cell-like and morphologically similar to the cells of the first stage except for the presence of a large nucleolus and numerous Golgi complexes. After 48 h from sphere dissociating (stage 6), SCs became electron-dark and resembled the SCs of the second stage by the presence of irregularly shaped nuclei and the cetoplasm filled with RER. We interpreted the results as senescence of the SCs with the number of passages after isolation from tissue and a day after dissociation of the spheres and as rejuvenation of the SCs just after sphere dissociation. Further research is needed to reveal the genetic, biochemical and physiological parameters of the SCs on established morphologically distinct stages in order to provide higher-quality cellular material for disease cell therapy.