Research Paper Volume 5, Issue 5 pp 357—372

Figure 6. hESC-secreted Factors Enhance NPC Proliferation and are Neuroprotective.

(A) Rat Neural Progenitor Cells (rNPCs) were cultured overnight in 50% differentiation medium (DMF12 + N2) and 50% specified medium followed by a 4 hour BrdU pulse to label proliferating cells prior to fixation. Immunofluorescence was performed for Sox2 (red) and BrdU (green), with Hoechst (blue) labeling cell nuclei. Representative images are shown. (B) Quantification of BrdU+/Sox2+ proliferating cells was performed by cell scoring in 100 random fields of each condition using an automated imager and MetaXpress cell scoring software. Results are displayed as the mean percent of BrdU+/Sox2+ proliferating cells +/−SD; N=4, *P< 2×10⁻¹⁵ for rNPCs incubated in hESC-conditioned Opti-MEM as compared to rNPCs incubated in differentiated hESC-conditioned Opti-MEM, and *P< 0.0002 for rNPCs incubated in hESC-conditioned Opti-MEM as compared to rNPCs incubated in Opti-MEM. (C) Pre-incubation of Aβ globulomers with hESC -conditioned Opti-MEM before incubation with mature cortical neurons prevents neuron cell death and exhibits a neurotrophic effect, as shown via decreased immunofluorescence staining of cleaved caspase3 (red) and increased Map2 + (green) neurons. Hoechst (blue) labels all nuclei. Representative images are shown. (D) Total number of Map2+ neurons and the amount of apoptosis was quantified by cell scoring of random fields taken by an automated imager of each condition in the above assay performed in replicates. Results are displayed as the mean percent of caspase + or Map2+ (C) proliferating or differentiating cells +/−SD, respectively. N=4, *P< 0.02 for Map2+ cortical neurons treated with Aβ globulomers preincubated in hESC-conditioned Opti-MEM, as compared to treatment with Aβ globulomers in OptiMEM, and *P< 0.05 for the level of caspase3 in cortical neurons treated with Aβ globulomers preincubated in hESC-conditioned Opti-MEM, as compared to treatment with Aβ globulomers in Opti-MEM.