174 ISOLATION, CULTURE AND POTENTIAL USE OF THE PORCINE NEURAL AND EPIDERMAL STEM CELLS

Abstract

The mammalian brain and epidermis contain stem cells, so-called neural stem cells (NSC) and epidermal stem cells (EpSC). To achieve the full therapeutic potential of stem cells, appropriate animal models have to be used to establish the sequence of pathological changes and to test potential therapies to block these changes. In the following studies miniature pigs were used as a biomedical model. We isolated multipotent cells from brains of porcine fetuses for future use in allotransplantation experiments in the inbred miniature pig strain. Brain tissue from 40- and 80-day-old porcine fetuses was mechanically dissociated, and cells were cultured in serum-free F12/DMEM medium with B27 and N2 supplements, EGF and bFGF. In 3–5 days some cells divided and formed floating spheres that were dissociated to single cell suspension and formed secondary spheres in culture. At all time points tested, the spheres represented mixtures of undifferentiated cells stained with nestin and Ki-67 antibodies and already differentiated neurons (Tu-20, MAP2) and glia (GFAP). After being plated on laminin/fibronectin coated coverslips and cultured in medium containing 2% FBS or 1 μM retinoic acid, the spheres adhered to the surface, and flattened, and cells started to migrate out. After immunofluorescence staining with antibodies to neuronal markers Tu-20 and MAP2, glial marker GFAP and oligodendrocyte marker CNPase showed that all the three cell types were present among differentiated cells. The EpSC are characterized by a slow and unlimited proliferation rate and, therefore, they retain labelled precursors of DNA more extensively than other keratinocytes. The main pool of EpSC is located in the bulge region of the hair follicle root sheath. A new procedure to isolate porcine hair follicles including their root sheaths was developed. The keratinocytes that migrated from hair follicles in the presence of feeder cells were poorly differentiated and specifically expressed galectin-1 or galectin-1-binding sites in their nuclei in co-localization with ΔNp63α. The exclusion of feeder cells from experimental system induced formation of spheroid bodies from these keratinocytes. Approximately one-third of these spheroids were able to adhere to a surface precolonized with feeder cells and to start forming normally growing colonies. Porcine hair follicles represent an excellent model for study of the functional phenotype of hair follicle-originated keratinocytes, and the endogenous lectin Gal-1 seems to be a potential marker of the porcine stem cell compartment of the hair follicle under in vitro conditions.