Fig. 9

Influence of cytokines on proliferation and differentiation of hypothalamic-derived embryonic NPCs grown in a neurosphere assay. a Cartoon depicting the neurosphere proliferation assay. NPCs isolated from the E12.5 embryonic mouse hypothalamus, plated, and left to proliferate. Neurospheres were counted (primary) from plating of 5000 cells/well. These cells were passaged at 1000 cells/well and left to proliferate into secondary neurospheres, or growth factors were removed to allow differentiation into neural lineages. Neurosphere assay of hypothalamic NPCs exposed to cytokines CCL2, CCL3, CCL4, CXCL2, or CXCL10 and assessed for alterations in proliferation ability when compared to control. b Primary neurosphere counts from direct plating of embryonic hypothalamic NPCs (ANOVA, P = 0.0464). c Secondary neurosphere counts following passage from primary neurospheres (ANOVA, P = 0.0147). Differentiation assay of hypothalamic neurospheres exposed to CCL2 and CXCL10 then assessed for differentiation into d NeuN+ neurons (P = 0.023 for CCL2, P = 0.048 for CXCL10), e PdgfRα+ OPCs (P = 0.022 for CCL2), and f Aldh1L1+ astrocytes (P = 0.44 for CCL2, P = 0.041 for CXCL10) when compared to control samples. Samples: Values are represented as number of neurospheres per 5000 cells plated for primary and per 1000 cells plated for secondary (n = 5 wells each for control and cytokines extracted from n = 5 independent pools of embryonic mouse brains). Values were normalized as fold-change to control and log transformed. Graphs: Box and Whisker plots with middle line representing median, cross representing mean, box extending from the 25th to 75th percentiles, and whiskers at max and min values. Statistics: ANOVA with Dunnet post hoc testing. *P ≤ 0.05