Figure 4.

STMN2 mis-splicing can be rescued in 2D

(A) Diagram showing the process by which 2D cultures are made. Briefly, iPSCs are differentiated into mature cortical-like astrocytes and neurons and assembled in a 1:1 ratio in 2D co-cultures. The co-cultures are grown for 4 weeks and then analyzed.

(B) Representative ICC images of 2D cultures stained for TDP-43, pTDP-43, MAP2, and DAPI (scale bar, 10 μm).

(C) Brightfield image of 2D cultures (GRN^+/+, GRN^−/−) at the 4-week timepoint (scale bar, 100 μm).

(D) Western blot of 2D co-cultures whole lysate of pTDP-43 and total TDP-43 in GRN^+/+ and GRN^−/− 2D co-cultures.

(E) Western blot quantification showing similar expression on pTDP-43 in GRN^−/− compared with GRN^+/+ 2D co-cultures when normalized to total TDP-43 (n = 4, unpaired t test, two tailed, p > 0.05, each n represents approximately 1 × 10⁶ cells and was repeated independently four times).

(F) Representative ICC images of 2D cultures stained for PGRN and DAPI showing positive staining in both GRN^+/+ and PGRN treated GRN^−/−but no PGRN staining in GRN^−/− co-cultures (scale bar, 10 μm).

(G) Quantification of CrSTMN2 expression using qPCR showing significantly higher expression in GRN^−/− compared with GRN^+/+ 2D co-cultures. The difference is rescued when GRN^−/− are treated with PGRN for 4 weeks (n = 10, one-way ANOVA followed by multiple comparison, ^∗p < 0.05, ns p > 0.05, each n represents approximately 1 × 10⁶ cells and was repeated independently three times). For all graphs, data are presented as mean ± standard error of the mean.