These results indicate that NSCs can be generated in defined conditions from hESCs adapted to defined conditions.
We therefore tested whether NSCs grown to passages 15 25 in defined conditions could differentiate into dopaminergic neurons in PA6-CM.
In fact, in order to derive "pure tumor" cell lines in defined conditions, all earlier approaches took measures to minimize the "contamination" of cultures with TME-elements.
In pursuing this goal, we have developed a GMP-compliant scalable process of generating neural stem cells (NSCs) and further differentiate them into dopaminergic neurons in defined conditions from hESCs adapted to a defined medium.
As seen in Fig. 2A, NSCs propagated in defined conditions for 15 passages expressed Sox1 and nestin, and differentiated into β-III-tubulin+ neurons, GFAP+ astrocytes and GalC+ oligodendrocytes in conditions promoting differentiation (Fig. 2B C).
We have previously shown that hESCs can be maintained in defined conditions for prolonged time periods while retaining their pluripotency, and our current experiments suggested that each of the remaining steps could utilize such a line to generate dopaminergic neurons.
Undifferentiated mES cells do not express Olig2 as determined by semi-quantitative RT-PCR (Figure 3A) but can be induced, in defined conditions, upon addition of FGF2 or with combinatorial application of FGF2, retinoic acid and Shh during directed differentiation of mES cells to motor neurons (Figure 3A) [35], [35].
Subsequent culture in defined conditions allows terminal differentiation of EBs to a specific cell type.
Furthermore, levels of Nanog are low in defined conditions but are comparable to those in ESCs for EpiSCs on feeders.
Culturing of CPCs in defined conditions for 14 days has resulted in near to normal functionality of these cells.
