Figure 4: Hippocampal marker-positive pyramidal and granule-like neurons were generated by long-term dissociation culture.

(a–c) Immunostaining of dissociated cells at day 197. Foxg1::Venus⁺ neurons (a) were connected to one another by MAP2⁺ neurites (b,c). GFAP⁺ glial-like cells were also detected (d). (e,f) KA1⁺/Foxg1::Venus⁺ neurons (CA type) tended to have large cell bodies; in contrast, Prox1⁺/Foxg1::Venus⁺ neurons (DG type) mostly had small somata (f is enlarged box in e). Zbtb20 was co-expressed with both KA1⁺ and Prox1⁺ neurons (g,h). (i) The percentage of Zbtb20⁺ cells was about 75% (means: 76.5%, s.e.m.: 1.65) and the percentages of KA1⁺ (means: 34.4%, s.e.m.: 1.83) and Prox1⁺ (means: 33.8%, s.e.m.: 2.33) were both about 34%. Countered neurons: Zbtb20⁺ (n=2764), KA1⁺ (n=147), Prox1⁺ (n=188). (j) Cell body size was significantly larger in KA1⁺ cells. ***P<0.001, paired t-test. Countered neurons: KA1⁺ (n=56), Prox1⁺ (n=55). (k,l) CaMKII was expressed in KA1⁺ (k) and Prox1⁺ (l) neurons. Scale bars, 200 μm (a–c); 50 μm (d); 20 μm (e,g–h); 10 μm (f,k,l). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).