The monolayer–spheres–monolayer cycle: Ultrastructural changes in stem cells

Аннотация

Stem cell sphere formation is applied as a preconditioning treatment prior to transplantation. Here, stem cells (SC) isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer–spheres–monolayer cycle by transmission electron microscopy. Spheres obtained on a non-adherent surface or through hanging drops gave similar results. At the first two or three passages (stage 1), isolated SC displayed an embryonal cell-like ultrastructure. With increased passage number (stage 2), SC became larger and more electron-dense with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), well-developed Golgi apparatus, and numerous vacuoles. At 2 h after sphere induction (stage 3), SC gathered into clusters and formed desmosome-like intercellular contacts. Their nuclei possessed a large loose fibrillo-granular nucleolus, the cytoplasm was tightly packed with disintegrated cisternae of RER, and Golgi apparatus was not identified. Twenty-four hours afterward (stage 4), SC in well-formed spheres exhibited a large dense nucleolus and poorly developed Golgi apparatus and RER. One day after sphere dissociation (stage 5), SC looked like embryonal cells and were morphologically similar to the cells of the first stage except for the presence of a large nucleolus and several Golgi complexes. At 48 h after sphere dissociation (stage 6), SC became electron-dense and resembled SC of the second stage, bearing irregular nuclei and cytoplasm that was rich in RER. We interpret these results as SC senescence with increasing passage number after isolation from the tissue or 1 day after sphere dissociation and rejuvenation of the SC just after sphere dissociation. Further research is needed to determine the genetic, biochemical, and physiological parameters of the SC corresponding to morphologically defined distinct stages in order to provide high quality cellular material for cell therapy.