Figure 4

Co-culture of ES-NS with LM-MP preparations for 10 days leads to a significant increase in neuronal differentiation. Expression of neuronal and glial markers was assessed by RT-PCR (A), Western Blot (B) and immunohistochemistry (C). The pan neuronal markers βIII-tubulin and PGP9.5 and peripherin, as well as the neuronal subtype marker nNOS were increased both at the mRNA and protein levels in the LM-MP co-cultured group as compared to control (fold change ranging from 1.3 to 3.2). The glial marker GFAP was not detected at this stage of differentiation by any of the analysis performed. (A) Changes of mRNA level in ES-NS co-cultured with LM-MP as compared to control cultures (ES-NS without LM-MP). n = 7. * P < 0.05, ** P < 0.01. (B) Changes in protein level in ES-NS co-cultured with LM-MP as compared to control cultures. n = 6. * P < 0.05, ** P < 0.01. (C) Changes in percentage of positive cells in ES-NS co-cultured with LM-MP as compared to control cultures. n = 3. * P < 0.05, ** P < 0.01. (D-E) Representative images of immunoreactivity for PGP9.5 (green), a marker for mature neurons, in control cultures and ES-NS co-cultured with LM-MP. PGP9.5 immunoreactivity extends to the neurites (arrows, E) although it is less intense partly due to the difference in focal planes. Scale bar = 25 μm. (F-G) Representative images of immunoreactivity for nNOS (red) in control cultures and ES-NS co-cultured with LM-MP showing increased proportion of nNOS⁺ cells after co-culture with LM-MP compared to controls. Scale bar = 50 μm. Nuclei are counterstained with DAPI (blue).