On ICC characterization of early EBs (5 days of culture in hanging drops, n = 5), only one EB expressed the ectodermal marker, vi-mentin, and none expressed cardiac troponin 1 as a mesodermal marker or a-fetoprotein, an endodermal marker. In an effort to allow more extensive differentiation, simple EBs were maintained in culture for up to 13 days, resulting in many of them (42-76%) progressing to a more complex structure containing a dark central core and a visible cavity (complex/cystic EBs) (Fig. 3B). In contrast to early EBs, all complex/cystic EBs (total of 15 days of culture in hanging drops, n = 7) expressed all three of the germ layer markers (Fig. 4, A-C). The NPC markers, nestin (Fig. 4D) and musashi1 (Fig. 4E), and a neuronal cell marker, neuron-specific enolase (Fig. 4F), were also detected in all cystic/ complex EBs. cialis canadian pharmacy online
Neural Progenitor Cells
The next step in differentiation was serum removal and culture in the presence of ITSFn medium. On culture of early EBs in ITSFn medium, a distinctive pattern of cells with varied morphologies emerged that was indicative of differentiation: tightly packed epithelial-like and fibroblastlike cells at the periphery of the outgrowth and small elongated cells in the center By Days 5-7, tubular structures of tightly packed columnar cells appeared in these EB outgrowths (Fig. 5A), which proliferated on addition of FGF-2 to the culture medium.