Figure 4

Co-culture of NSC-MN spheroids and HUVEC in collagen gel on a Petri dish and neuronal activity with and without HUVEC networks. (a) After 2 days of co-culture with HUVEC and MN spheroids, HUVEC formed well-connected microvascular networks around the spheroid. Almost all microvascular networks exist surrounding MN spheroids, but a few HUVECs invaded into the MN spheroids and formed networks inside. After 3, 4 days, MN spheroids start to migrate out of the initial spheroid and penetrate into the surrounding collagen gel, resulting in the formation of long neurites. (b) Immunostaining of actin, Tuj1 and DAPI show that vascular networks are formed prior to neurite elongation by Day 3. Neurite elongation is observed during the next several days. (c) After 7 days in culture, interpenetrating MN networks and vascular networks can be observed. MN neurites directly attach to the vascular networks enabling communication between them. (d) HUVEC networks promote neurite elongation of MNs. n = 3; *P < 0.01, two-way ANOVA. (e) Quantification of maximum neurite length. (f) Spontaneous Ca²⁺ oscillation by fluo-8am. EC networks increase the frequency, amplitude, and synchrony of neuron activity. (g,h) The concentration of BDNF and BMP2 in culture medium with and without HUVEC networks. HUVECs secreted BDNF which can upregulate neuronal differentiation and synapse formation and promote neuroprotection. In contrast, the concentration of BMP2 did not significantly increase to affect cellular activity. n = 3; *P < 0.01, two-way ANOVA. (i) Relative change of mRNA expression related to juxtacrine signaling. Notch1 and DLL4 are both significantly increased, resulting in synaptic stabilization. n = 3; *P < 0.01, two-way ANOVA. All error bars ± SD.