Total RNA or cell lysate was prepared from cells at the hESC, NES, isolated rosette, R-NSC, NPC monolayer, Neural I, or Neural II stages for quantitative RT-PCR (A) or immunoblot analysis (B). Arrowhead indicates endogenous MEF2C. Anti-PSD95 antibody was used to assess neuronal differentiation. Histogram values are mean + SEM, n = 3; *p<0.001 compared to hESC by ANOVA.

(A) Lentiviral-infected cells plated on poly-l-ornithine/laminin. Anti-GFP antibody identifies infected cells and anti-DCX antibody identifies early neurons. Scale bars: 25 µm. (B) Neuronal enrichment engendered by MEF2CA (n = 4 experiments; scheme for infection and analysis shown in Figure S3D). In each experiment, ∼300 GFP-positive cells were scored. Values are mean + SEM, n≥1200 cells counted; ***p<0.001 by ANOVA. (C) Longest neuronal process per cell at 33 dpi during Neural Stage II, measured with Neuron J software. Values are mean + SEM, n = 61; ***p<0.0001 by ANOVA. (D) Neuronal-specific proteins in lenti-MEF2CA-infected cells by immunoblot during Neural Stage II. Note the much stronger GFP expression in control cells because GFP was expressed from a single gene construct rather than in tandem with MEF2CA and IRES [53]. MEF2CA increased expression of neuronal proteins compared to control infection. (E) Whole-cell recordings of lenti-MEF2CA-infected cells with patch electrodes revealed sodium currents evoked by 100 ms depolarizing steps from −60 to +80 mV in 20 mV increments following 300 ms prepulse to −90 mV (n = 5); these currents were inhibited by tetrodotoxin (TTX). (F) Application of 10 pA current steps resulted in depolarization and generation of a “train” of action potentials during current-clamp recordings in n = 3 of 8 (37.5%) MEF2CA-infected cells. (G, H) GABA-evoked currents were observed in n = 5 of 7 cells (71.4%) and NMDA-evoked currents in n = 3 of 3 cells recorded (100%).

(A) Infection/transplantation scheme (cf. Fig. 1A). (B) Apomorphine-induced rotations in 6-OHDA-lesionsed rats after R-NSC transplantation into the striatum. Values are mean + SEM, n = 14. Rats receiving MEF2CA/R-NSCs show increasing improvement versus controls (*p≤0.035 by ANOVA with planned comparisons post-hoc test). (C) Cylinder asymmetry test 9 weeks post-transplant. Forelimb use in transparent cylinder for 10 min. For score calculation, see Text S1. Values are mean + SEM, n = 11; *p<0.03 by t-test. (D) Transplanted control R-NSCs (GFP+/green); sagittal brain sections along mediolateral axis. Hoechst dye-stained DNA (blue). Scale bar: 1 mm. Inset: Hematoxylin/eosin (H&E) stain shows xenograft/host boundary (dashed line). Xeno: xenograft; STR: striatum; LV: lateral ventricle. (E) Human origin of cells verified by human nuclear antigen (HNA). Scale bar: 25 µm. (F) Infected cells (GFP+) co-expressing HNA (red) to yield merged yellow fluorescence (remaining GFP positivity represents cell debris). Scale bar: 25 µm. (G) Density of TH+ neurons in MEF2CA/R-NSC xenografts (xeno) close to the ventral area of the striatum was higher than for control/R-NSCs (boxed areas enlarged in right panels). Arrowheads: TH+ neuronal cell bodies; arrows: neuronal processes; dashed lines: graft/host boundary based on H&E staining; blue dashed box: outlines endogenous host TH+ fibers (quantified in Figure S7). (H) MEF2CA/R-NSC versus control/R-NSC DA/TH+ neurons. Transplanted cells are GFP+; TH+ cells are red. Scale bar: 25 µm. (I) TH+ neurons in grafts as percent of GFP+ cells (upper panel). Density of TH+/GFP+ cells in grafts as absolute cell number (lower panel). Values are mean + SEM, n = 19; ***p<0.0001 by t-test.

(A) Phase-contrast images were taken during various stages of differentiation in vitro and represent cell morphology of each developmental step. Arrows indicate rosettes that formed on laminin-coated dishes. hESC, human embryonic stem cell; NES, neuroectodermal sphere; R-NSC, rosette-neural stem cell; NPC, neural progenitor cell (see Materials and Methods). (B) Representative cells from the NES, NPC, Neural I, Neural II, and Neural III stages of development were fixed and stained with the following lineage-specific antibodies. For NPCs: Msi1 (musashi 1) and Nestin. For neurons: DCX, MAP2, NeuN, Synp (synaptophysin), and PSD95. For astrocytes: S100β. For oligodendrocytes: CNPase. DNA stained with DAPI (blue). Scale bar: 25 µm.

(A) TUNEL assay on NPCs at 14 dpi and on Neural Stage II cells at 33 dpi with control or lenti-shMEF2C shRNAs. Lenti-shMEF2C-1-infected cells show increased apoptotic cell death at 14 but not 33 dpi. Values are mean + SEM, n = 6; ***p<0.001 by ANOVA. dpi: days post infection. (B, C) Endogenous MEF2C and MAP2 expression at 33 dpi by qPCR and immunoblot. Values are mean + SEM, n = 3; **p<0.01, ***p<0.001 by ANOVA. (D) Fewer MAP2+ (red) neurons after lenti-MEF2C-1 shRNA versus scrambled shRNA (GFP+/green) at 33 dpi indicate absent or delayed neurogenesis at Neural Stage II. (E) Percentage of MEF2C shRNA-transduced R-NSCs (total GFP+ cells) differentiated into neurons (MAP2+/GFP+ cells). Values are mean + SEM, n≥140 cells scored for each condition; **p<0.01, ***p<0.001 by ANOVA. (F) Shorter dendrites, fewer dendritic spines in lenti-shMEF2C-1-infected cells versus scrambled control. shRNA-infected cells are GFP+ (green), neuronal dendrites are MAP2+ (red); co-labeling (yellow) indicated by asterisks; dendrites in boxes magnified to show spines. DAPI (blue). Scale bars: 25 µm. (G) Dendritic length using Neuron J software (dendrites scored if ≥2-fold longer than diameter of cell body). Values are mean + SEM, n = 32 cells; **p = 0.003 by t-test. (H) Numbers of dendritic spines for lenti-MEF2C-1 shRNA-transduced neurons and scrambled control (spines scored along entire length of dendrite). Values are mean + SEM, n = 17 cells; ***p<0.0001 by t-test.

(A) hESC-derived cells at Neural Stage III stained for MAP2 (red/neurons), tyrosine hydroxylase (TH/green, DA phenotype; lower panel magnified). Some TH+ cells did not express MAP2 [54]. Scale bars: 25 µm. (B) Relative mRNA levels of DA-specific genes at various stages. Values: mean + SEM, n = 3; *p<0.05 by ANOVA. EN1, engrailed-1. (C) EN1+ or TH+ cells expressed VMAT2 (vesicle monoamine transporter 2) and DAT (dopamine transporter). Scale bars: 25 µm. (D) Expression of GIRK2/DAT+ and CD28k/TH+ cells. (E) Control vector- and MEF2CA-infected cells (GFP+/green) stained for TH (red, left-hand panels) or EN1 (red, right-hand panels), with double-labeled cells (yellow) predominating in the MEF2CA group. Scale bars: 50 µm for TH; 25 µm for EN1. (F) Percentage of MEF2CA/R-NSCs vs. control cells (GFP+) differentiated to DA neurons during Neural Stage I at 17 dpi (TH+, left graph) and Neural Stage II at 32 dpi (EN1+, right graph). Values: mean + SEM, n≥1200 TH cells, n≥500 EN1 cells; **p = 0.001, ***p = 0.0007 by t-test. (G) qPCR shows relative amount of DA gene expression during Neural Stage I at 13 dpi. Values: mean + SEM, n = 3; **p<0.01, ***p<0.001 by ANOVA; endo MEF2C: endogenous MEF2C. (H) qPCR shows expression of Etv1 at different stages. Values: mean + SEM, n = 3; **p<0.01 by ANOVA. (I) MEF2C activation of 2.1 kb Etv1 promoter-luciferase construct in HeLa cells. Values: mean + SEM, n = 6; *p<0.05, ***p<0.001 by ANOVA. MEF2DN: dominant negative MEF2C; MEF2C: full-length MEF2C; MEF2CA: constitutively active MEF2C. (J) ChIP analysis of MEF2C association with the Etv1 promoter. Values: mean + SEM, n = 3; ***p<0.001 by t-test.

(A, C) Twelve weeks after transplantation, infected (GFP+) cells were analyzed with cell-type specific antibodies (anti-HuC/D for neurons and anti-GFAP for astrocytes, although neural progenitors can also be labeled with this marker). (B, D) Quantification of neuronal and astrocytic markers in control- and MEF2CA-infected cells. Ten random fields were selected in multiple sections at the same distance from the Bregma for each rat (n = 4). Values are mean + SEM, n = 10; *p<0.03 by t-test. (E) In transplanted brains, several regions manifested rosette structures (asterisks), consistent with hyperproliferation. For control-infected/R-NSC transplants (left-hand and middle panels), the GFP+ cells (arrows) were located within rosettes. MEF2CA/R-NSC transplants contained both MEF2CA-expressing cells (GFP+, green) as well as uninfected R-NSCs, but GFP+ cells (indicated by arrows) were located exclusively outside of the rosettes (right-hand panel). All scale bars: 25 µm.