Abstract:
The discovery of endogenous neural progenitor cells (NPCs) in the adult human brain capable of migrating and differentiating in response to external stimuli has re-defined the field of neuroscience. In order to study and characterize these NPCs, research groups have attempted to culture them in vitro with varying success. Adult human NPCs are generally cultured from the two major neurogenic regions of the brain; the subventricular zone (SVZ) and the dentate gyrus of the hippocampus. However, we propose that cells with similar characteristics to NPCs can be isolated and cultured from other regions of the adult human brain. Briefly, human brain cells were isolated from the middle temporal gyrus (MTG) of post-mortem and biopsy tissue and diced into small pieces prior to enzymatic digestion. The dissociated cells were plated onto poly-L-lysine (PLL) coated culture flasks. Initially the cells were maintained in media consisting of 10% serum, DMEM:F12 media supplemented with 2mM glutamine and antibiotics at 5% CO2/37ºC. Once confluent, the cells were re-plated into conditions favourable for NPC growth (serum free Neurobasal medium supplemented with N2, 2mM GlutaMAX® and antibiotics or commercially available STEMPRO® medium (Invitrogen)) in either adherent monolayer cultures or non-adherent surfaces for the formation of spheres. When cells from the MTG were cultured in 10% serum, the dominating cell type that arose after prolonged culture periods were fibroblast-like cells (FbCs), which were highly proliferative and expressed high levels of fibroblast markers. However, when cultured under NPC conditions, FbCs became bipolar in morphology and strongly expressed genes associated with NPCs such as nestin, GFAP and GFAP-δ and βIII-tubulin. These expression patterns partially resembled those seen with adherent adult human NPCs isolated from the SVZ. Furthermore, when FbCs were cultured on non-adhesive surfaces, they formed spherical structures resembling neurospheres formed from adult human SVZ-derived NPCs. When these FbC-spheres were plated onto adhesive surfaces they gave rise to cells expressing immature neuronal markers. Although further investigation is needed, these results suggest that more than one cell type from the adult human brain in vitro possesses stem cell-like properties. The origin of these FbCs is yet to be determined, however, leptomeningeal or blood-born mesenchymal precursor (fibrocytes) origin cannot be excluded. Regardless of their origin, these cells provide further evidence for cellular plasticity and possibly provide an alternative in vitro source of adult neural stem cells.