Fig. 2. Identification and In Vitro differentiation of PD AHNPs.

Immunocytochemistry was utilized to establish PD AHNPs as neural stem/progenitor cells (A–F from SN cultures). Cells in expanding cultures express neural stem cell markers nestin (A; green) and SOX2 (Brazel et al., 2005; E; red), and the majority of the nestin/SOX2 positive cells are also immunoreactive for radial glial markers vimentin (C; red) and GLAST (EAAT1) (F; green). (B) GFAP (green) expression in a subpopulation within these primary cultures. (M) There is no significant difference (p=0.47, 0.49) in the proportions of nestin and SOX2 positive cells within AHNP populations from the four brain regions studied here (SN, SVZ, HC, CX). RT-PCR was performed on SN-derived cells and SVZ and SN tissue to detect mRNA for progenitor cell markers. Both cell (N) and tissue (O) RT-PCR results show nestin, SOX2, vimentin and GLAST expression, suggesting a progenitor cell pool in the PD patients’ SN and SVZ. The expression of GLAST in SN tissue is markedly weaker than that seen in SVZ tissue. Under appropriate differentiation-inducing conditions, PD AHNPs have been shown to have the capacity to differentiate into both neurons and astrocytes, revealing their multipotent neurogenic potential (G–L images were taken from human PD SN AHNPs and mouse GFP-ESNPs co-cultures). Co-cultured with GFP expressing mouse ESNPs in DM containing SHH, FGF8b, BDNF, NT-3, PDGF, ascorbic acid and retinoid acid, PD SN AHNPs (not GFP+) gave rise to TuJ1 positive neurons (G,H,I; labeled with white arrow, red is Tuj1), TH positive DA neurons (J,K; labeled with white arrow, red is TH) and GFAP positive astrocytes (L; red is GFAP). GFP green immunofluorescence in (G–L). Nuclei are counterstained with DAPI (blue). (Scale bars = 50 μm)