Fig. 4

Effect of myocyte-conditioned media on neural and endothelial cell cultures. A)Endothelial cells (HUVEC) were cultured as a monolayer in various media formulations to study the effect of myocyte conditioned or motor neuron-myocyte coculture conditioned media on endothelial cell survival and growth through expression of CD31 marker (purple). Nonconditioned triculture media was found to be not conducive for endothelial cell growth. Media conditioned with either myocytes only or myocyte-motor neuron coculture was observed to promote endothelial cell survival and growth like the recommended media for HUVEC (vascular media) used here as a positive control. Scale bar — 250 µm. B Myocyte-conditioned triculture media was used to maintain a coculture of motor neurons and endothelial cells for 14 div to study the effect of myocyte conditioning on neurovascular health. Motor neurons and endothelial cells improved cell morphology and growth as evidenced by expression of mature motor neuron markers (ChAT and SMI-35) and endothelial marker (CD31), when cultured in myocyte-conditioned triculture media. Nonconditioned triculture media was found to be not supportive of neuronal or endothelial cell growth. Scale bar — 250 µm