Unlike clonal cultures, these neurospheres were formed by aggregation, which results in culture heterogeneity.The minor transgene expression variability among Lomitapide Mesylate neurosphere cultures generated from littermate fetuses possibly occurred during the initial brain harvest. We did not specifically dissect the forebrain from each fetus. Nestin expressing neural SCs in the developing midbrain and hindbrain may have contributed to the transgene expressing cell population since CKIIa, the promoter driving tTA-transgenic human tau expression, is expressed throughout the brain at this developmental age. We saw more variability among independent experimental harvests than among cultures derived from littermate fetuses; we attribute this variation to inconsistencies inherent to IFA. Regardless, IFA consistently demonstrated that undifferentiated cells derived from rTg expressing fetal brains expressed human tau in a higher proportion of cells than those derived from rTg expressing fetal brains. Total brain homogenates Mechlorethamine hydrochloride indicate that rTg mice express comparable levels of transgenic tau as rTg. While the rTg cultures had a lower proportion of total cells expressing human tau, they contained a greater proportion of cells that expressed higher levels of tau. This feature of some transgenes is caused by position-effect variegation. Neurospheres, like the mice from which they were derived, may show this effect and could be useful models for screening transgene expression in founder lines. Consistent with the difference in transgene expression seen in undifferentiated neurospheres, differentiated cells derived from tauwt-expressing neurospheres expressed human tau in a higher proportion of cells than those derived from tauP301L-expressing neurospheres. Likely, non-transgene expressing progenitor cells gave rise to nontransgene expressing differentiated cells and transgene expressing progenitor cells differentiated into transgene expressing mature cells. Alternatively, tauP301L transgene expression may have decreased neural precursor survival. Since neurospheres proliferated and differentiated over several passages in both genotypes, and differentiated transgene expressing cell proportions mirrored that of the undifferentiated condition, evidence favors the former explanation. Some of the differences we saw between tauP301L and tauwt may stem from differences in transgene insertion sites. Neurosphere cultures from mouse lines expressing the same human tau variants, but at lower levels than rTg4510 and rTg21221, overlapped in the percentages of cells expressing human tau, but within the same range as our more extensively studies lines. It is likely that there are effects of transgene insertion site as well as the tau mutation. With the emergence of methodologies to culture neurospheres from human patients, experiments evaluating the culture system’s relevance and validity are crucial. Our data provide supporting evidence that neurospheres can reliably model phenotypes of their derived source over extended culture periods. The neurosphere culture system provides a robust assay for studying effects of external factors 
on the development and differentiation of the CNS, and the genetic susceptibility to neurological disorders. While it is unreasonable to expect that an in vitro system will fully recapitulate a complex disease process involving complex cell interactions, biologically relevant models that provide reproducible results are invaluable resources.