Figure 2. Direct Conversion of Young and Old Human Fibroblasts into Functional iNs Is Efficient Regardless of Donor Age.

(A) Direct iN conversion of young and old donor fibroblasts using Ngn2-2A-Ascl1 (N2A) and small molecular enhancers (SM).

(B) Schematic of lentiviral system for inducible overexpression of N2A and SM for iN conversion.

(C) Phase contrast images of progressively converting fibroblasts, and immunofluorescence images of 6-week converted fibroblasts stained with βIII-tubulin, hTau, NeuN, Map2ab, and vGlut1.

(D) iNs co-cultured on astrocytes labeled with LV-hSyn∷RFP and stained for synapsin-I following 8 weeks of conversion.

(E) Electrophysiological characterization of iNs shows multiple evoked and spontaneous action potentials.

(F) Immunocytochemical characterization of iN from young and old donors after 3 weeks of conversion. All of the scale bars represent 20 μM.

(G) Quantification of neuronal yields per DAPI from 18 donors following 3 weeks of conversion. The bar graphs show mean + SEM.

(H) Electrophysiological characterization of young- and old-derived iNs shows Na⁺/K⁺ channel-mediated inward/outward currents in response to depolarizing voltage steps (upper) and multiple evoked action potentials (lower) without apparent differences between young and old (n = 13 donors).

(I) Quantification of neuronal subtype markers based on immunocytochemical analysis (extended data in Figure S2).

Also see related Figure S1.