The main finding of this study is that ECs modify the proliferation of hES cell-derived, human NPCs and their development of electrophysiological neuronal properties. When Sox-2- and nestin-expressing NPCs were cultured in the presence of FGF-2, they proliferated and showed only outward membrane currents. Removal of FGF-2 led to biochemical differentiation, manifested by the expression of βIII-tubulin, DCX, Hu, calbindin, and GABA by 10 days, and electrophysiological differentiation, evidenced by the acquisition of a small inward current with a reversal potential close to the equilibrium potential for Na+, by 14 days. NPCs co-cultured with ECs showed a similar time course of neuronal marker protein expression, but accelerated development of inward current, which was detectable 5 days after removal of ECs and withdrawal of FGF-2, and which was larger in magnitude at 14 days than the inward current in NPCs cultured without ECs. In addition, action potentials were seen more frequently at 14 days in NPC-EC co-cultures. We conclude that ECs enhance cell proliferation and hasten the development of electrical but not biochemical neuronal properties in NPC cultures, and that the effect of ECs is likely to depend on diffusible mediators rather than cell–cell contact, because NPCs and ECs in co-culture were separated by a barrier that precluded transmigration.
Several prior studies have documented the transition of NPCs to more functionally mature cells of neuronal lineage in vitro. In rodents, cultured ES cells [19
] and NPCs from embryonic [21
], fetal [23
], or adult [24
] brain develop inward and outward membrane currents and action potentials consistent with the expression of voltage-gated Na+
channels. Similar observations have been made for NPCs cultured from embryonic [26
] or adult [27
] human brain. The electrophysiological properties of undifferentiated hES cells and hES cell-derived dopaminergic neurons maintained in culture have also been described. Undifferentiated hES cells showed depolarization-activated, tetraethylammonium-sensitive delayed rectifier K+
currents, but neither Na+
]. In contrast, hES cells induced to undergo dopaminergic differentiation by co-culture with stromal cells that overexpress sonic hedgehog developed fast inward currents and delayed outward currents with slight inactivation, rapidly inactivating A-type currents and action potentials [31
As noted previously, Shen et al. [12
] reported that the in vitro proliferation of murine NPCs could be enhanced by co-culture with ECs, providing a possible mechanistic basis for the neurovascular stem-cell niche [9
]. When Transwell inserts containing primary bovine pulmonary or clonal mouse brain ECs were added to mouse embryonic cortical cell cultures, the latter expanded more rapidly, but the percentage of cells expressing the neuronal marker β
III-tubulin was lower. Removal of the inserts was associated with an increase in the percentage of cortical cells that went on to express the neuronal marker β
III-tubulin, compared to pure cortical cultures, or to cortical cells co-cultured with fibroblasts or vascular smooth muscle cells, without change in the percentage of GFAP+
astrocytes. The authors concluded that endothelium-derived factors stimulate the proliferation of NPCs and inhibit neuronal differentiation. Our findings are substantially in agreement, in that co-culture of ECs with hES cell-derived NPCs led to an increase in cell proliferation but not in biochemical neuronal differentiation, as determined by marker protein expression. One difference between the two studies is that we withdrew FGF-2 from NPC cultures when EC-containing Transwell inserts were removed, whereas Shen et al. [12
] did not. Therefore, the differences that we observed between control and EC-treated NPC cultures could depend on concurrent FGF-2 withdrawal. Another difference is that we studied not only protein marker expression, but also the development of electrical neuronal properties. These studies revealed a possible role of ECs in promoting the electrical, as opposed to biochemical, neuronal differentiation of NPCs. Alternatively, the sole effect of ECs could be to increase NPC proliferation, and the more prominent TTX-sensitive currents in EC-treated cultures could simply reflect the presence of more neurons.
The induction of electrical neuronal properties in hES cell-derived NPCs by other cell types has also been described. Co-culture with astrocytes from neonatal or adult hippocampus, or culture on astrocyte-conditioned substrates, promoted the development of TTX-sensitive action potentials in adult rat hippocampal NPCs [24
]. In another study, Johnson et al. [32
] followed the development of neuronal electrical properties in cultured hES cells induced to undergo neuronal differentiation in the absence of FGF-2 and the presence of brain- and glial-derived neurotrophic factors. Neuronal differentiation was accompanied by the emergence and subsequent maturation of TTX-sensitive Na+
action potentials, which showed progressively increased amplitude and decreased duration. Co-culture of hES cell-derived NPCs with astrocytes had little effect on cell-intrinsic neuronal properties, but accelerated the development of synaptic activity, manifested by spontaneous, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABA receptor-mediated postsynaptic currents. In contrast to our results and those of Shen at al. [12
] regarding ECs, the effect of astrocytes appeared to depend on cell–cell contact, because it was abolished if NPCs and astrocytes were plated on opposite sides of a Transwell insert.
The stem-cell niche in which neurogenesis occurs comprises a variety of cell types and extracellular components, which contribute to the production of neurons through diverse mechanisms. In addition to NPCs, which are the direct predecessors of new neurons, these include cells that regulate neuronal differentiation. This study and previous work cited above [12
] suggest important roles for ECs and astrocytes in this latter process. In particular, soluble factors released from ECs contribute to the expression of ion channels underlying neuroexcitability, whereas contact with astrocytes helps guide the subsequent development of transsynaptic neuronal activity. The roles of other constituents of the stem-cell niche, including vascular smooth muscle cells, pericytes, circulating blood cells, and extracellular matrix remain to be clarified, especially in the context of human neurogenesis.