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. 2020 Sep 24;15(4):869–882. doi: 10.1016/j.stemcr.2020.08.013

Figure 5.

Figure 5

Progressive Subtype-Specific Maturation of the iDANs over Time

(A, C, and E) The proportion of TH+ iDANs increase when doxycycline is withdrawn after 3 weeks and the ALN + shREST reprogrammed neurons are kept in culture for 50 days (C) and 100 days (E), compared with the 3-week time point (A).

(B, D, and F) The proportion of TH+ iDANs increases when doxycycline is withdrawn after 3 weeks and the ALN + Foxa2 + shREST reprogrammed neurons are kept in culture for 50 days (D) and 100 days (F), compared with the 3-week time point (B).

(G) Quantifications of neuronal proportion and TH+ neuronal proportion over time. TH+ neuronal proportion (ALN + shREST): 3 weeks, 3.6% ± 1.0%, n = 7; 50 days, 31.4% ± 5.6%, n = 2; 100 days, 53.9% ± 8.3%, n = 3. TH+ neuronal proportion (ALN + Foxa2 + shREST): 3 weeks, 1.8% ± 0.8%, n = 5; 50 days, 34.5% ± 3.6%, n = 2; 100 days, 50.2% ± 4.9%, n = 3. Neuronal proportion (ALN + shREST): 3 weeks, 41.8% ± 5.0%, n = 7; 50 days, 49.8% ± 18.1%, n = 2; 100 days, 45.8% ± 8.0%, n = 3. Neuronal proportion (ALN + Foxa2 + shREST): 3 weeks, 34.3% ± 5.0%, n = 5; 39.2% ± 11.7%, n = 2; 100 days, 38.4% ± 14.2%, n = 3.

(H–J) The generated iDANs are positive for SYN1 (H), VMAT2 (I), and ALDH1A1 (J).

(K and L) TH+/DDC+ double-labeled cells are observed in long-term cultures of both ALN + shREST (K) and ALN + Foxa2 + shREST (L) reprogrammed cells. White arrows indicate examples of double-labeled cells.

Data are presented as means ± SEM and all data points have been visualized in the graphs. Each data point represents a replicate from an independent experiment (n = 7 for ALN + shREST at the 3-week time point; n = 5 for ALN + Foxa2 + shREST at the 3-week time point; n = 2 for both conditions at the D50 time point and n = 3 for both conditions at the D100 time point). In (G), the proportion of TAU+ and TAU+/TH+ cells at the 3-week time point was compared with the late time points day 50 and day 100 combined using an unpaired, two-tailed t test. ∗∗∗p < 0.001 (p < 0.0001 for TH+ cells in ALN + shREST, p < 0.0001 for TH+ cells in ALN + Foxa2 + shREST); n.s. (p = 0.5307 for TAU+ cells in ALN + shREST, p = 0.6637 for TAU+ cells in ALN + Foxa2 + shREST). Scale bars: 50 μm. See also Figure S6.