Figure 1.

GRN^−/− mbOrgs show features of human TDP-43 proteinopathy after 4 weeks in culture

(A) Diagram showing the process by which mbOrgs are differentiated and assembled. Briefly, iPSCs are differentiated into mature cortical-like astrocytes and neurons and assembled in a 1:1 ratio in 3D co-cultures called mbOrg. The mbOrg are grown for four weeks and then analyzed.

(B) Brightfield image of two mbOrgs (GRN^+/+, GRN^−/−) kept in culture for 4 weeks (scale bar, 100 μm).

(C) Confocal images of mbOrg slices (GRN^+/+ or GRN^−/−) showing expression of NF1A, TUJ, and DAPI after 4 weeks in culture (scale bar, 50 μm).

(D) Western blot of mbOrgs whole lysate showing higher expression of pTDP-43 in GRN^−/− compared with GRN^+/+ mbOrgs when normalized to total TDP-43.

(F) Western blot quantification showing significantly higher expression of pTDP-43 in GRN^−/− compared with GRN^+/+ mbOrgs when normalized to total TDP-43 (n = 4, unpaired t test, two tailed, p < 0.001, each n represents approximately 50 mbOrgs and was repeated independently four times).

(E) Confocal images of mbOrg slices (GRN^+/+ or GRN^−/−) showing expression of NFIA, TDP-43 and DAPIafter 4 weeks in culture (scale bar, 10 μm).

(G) Quantification of CrSTMN2 expression using qPCR showing significantly higher expression in GRN^−/− compared to GRN^+/+ mbOrgs (n = 11±, unpaired t test, two tailed, p ≤ 0.001, each n represents approximately 50 mbOrgs and the experiment was repeated independently four times).

(H) IMARIS 3D reconstruction of TDP-43 and pTDP-43 staining in 4-week-old mbOrgs (scale bar, 10 μm).

(I) quantification of extranuclear TDP-43 particle count per square millimeter, showing significantly higher count in GRN^−/− compared with GRN^+/+ mbOrgs. Each dot represents one independent mbOrg (GRN^+/+ n = 4, GRN^−/− n = 6, unpaired t test, two tailed, p < 0.05, each n is one mbOrg and the experiment was repeated independently three times). For all graphs, data are presented as mean ± standard error of the mean.