Figure 2. EdU⁺ cells have sphere-forming potential and express ENSC markers.

Following a “pulse” with EdU, LMMP was digested and cells were cultured in media without EdU for a prolonged “chase” (up to 25 days). After either 1 or 2 passages, dissociated cells were seeded into microwells allowing tracking of single cells. Using fluorescence microscopy, single tdT-expressing cells (A, arrow) were observed that formed NLBs (B, arrow). Upon fixation and labeling of EdU, subsets of NLBs were found that contained EdU⁺ cells (C, arrow). The majority of NLBs remained small (≤3 cells/sphere cross-section) and none of these contained EdU+ cells (D). However, EdU was detected in a substantial subset of large NLBs (>3 cells/sphere cross-section) (E). The average sphere size was larger in NLBs that EdU was detected, a difference that was statistically significant for both secondary and tertiary NLBs (F). Flow cytometric analysis was performed on LMMP that was pulsed with EdU at 48 h and gated for tdT⁺/EdU⁺ single cells with debris and doublets excluded. The >97% of these cells co-expressed ENSC markers (white histogram) including p75, CD49b and Nestin (D, gray histogram is no primary control). * = p<.0001 by t-test. Error bars indicate SEM. Scale bar: 100 µm in A and B, 25 µm in C, D, and E.