Figure 2.

Comprehensive classification of subependymal cells

(A) Defined flow cytometry panel with the specific combination of markers used to identify the different SEZ populations.

(B) Initial steps to select Lin^– cells after excluding cell aggregates and discarding CD45/O4/CD31/Ter119-positive and dead cells.

(C) Representative FACS plot showing GLAST and CD24 staining levels in the Lin^– fraction and segregation of the cells into four categories.

(D) Based on the expression of the activation marker EGFR, the GLAST^–CD24^–/low region defines EGFR⁺ NPC1 (left), the GLAST⁺CD24^high fraction corresponds to EGFR⁺ NPC2 (center) and the GLAST^–CD24^high region contains EGFR⁺ NB1 and EGFR^– NB2 (right).

(E) Among the GLAST⁺CD24^–/low fraction, CD9^high levels distinguish between GLAST⁺ NSCs from non-neurogenic GLAST⁺ astrocytes. CD9 plot for cortex astrocytes is adapted from Belenguer et al., 2021.

(F) GLAST and EGFR levels define three NSC populations: qNSC (GLAST^highEGFR^–/low), pNSC (GLAST^lowEGFR^–/low) and aNSC (GLAST^lowEGFR⁺).

(G) Estimated number of cells for each SEZ population considering an approximate total viable cell yield of 100,000 cells per brain.

(H and I) Representative FACS plots showing EdU staining in the Lin^– fraction following two different pulse and chase regimes described in Belenguer et al., 2021: 1 shot of EdU followed by 1 h chase (H) and 7 injections of EdU (q2 h: one every 2 h) followed by a 12 h chase (I). aNSC and NB2 populations are included as examples for these two regimes.