Fig 2: NSUN2 and Angiogenin Affect Neural Stem Cell Differentiation(A) Transfection of siRNA (NSUN2 and ctr), time course of NES cell differentiation, and sample collection.(B–D) NSUN2 (B) and TUBB3 (C, D) RNA expression at days 0, 4, and 8 after growth factor removal in the presence of an NSUN2 siRNA (B and C) or recombinant angiogenin (rANG) (D). Shown is ??Ct relative to GAPDH. Error bars denote SD (n = 3 transfections per time point).(E) Treatment with rANG, the siRNAs for NSUN2, or a scrambled RNA as control (Ctrl), and time course of NES cell differentiation.(F) Flow cytometry for NCAM and CD24 in control cells and cells treated with rANG and siRNAs (siNSUN2 or siCtrl) at day 0 (left-hand panels) and day 8 (right-hand panels) of the differentiation protocol.(G) Quantification of cells shown in (F). n = 5 experiments per condition. Error bars denote SD. ***p < 0.001, Student's t test.See also Figure S2.

Fig 3: NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C). (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 µm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 µm. (Gi) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (Gii). (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 108) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I), single stained with DiI (J), and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.

Fig 4: The 5-HT6 Receptor Is Highly Expressed in Human Tissue Grafts Originating from Both Fetal and hESC Sources(A–E) Immunofluorescence staining of 5-HT6 expression in a human dopaminergic fetal graft within the rat striatum (A and C) with the phenotypic staining for TH (A and B), and the identification of human NCAM (A and D), imaged using LSM. The 5-HT6 expression was confirmed to reside in DA neurons originating from the transplanted tissue (E).(F–J) Similarly, immunofluorescence staining of a paraffin-embedded section originating from the putamen of a PD patient that received a dopaminergic fetal graft, imaged using LSM, confirmed that the 5-HT6 receptor (F and H) is highly expressed in the TH-positive (F and G, with overlay in J) DA neurons originating from the fetal graft. Nuclear staining (DAPI) confirmed the 5-HT6 expression to be abundant in the neuronal soma (I).(K–O) To confirm if this receptor is also abundant in DA neurons differentiated from hESCs, a graft derived from the H9 hESC line, differentiated using the novel floor-plate-based protocol and transplanted to the parkinsonian striatum of a nude rat, was evaluated using LSM with immunofluorescence for the same genes: 5-HT6 (K and M), TH (K and L), and the human NCAM (K and N). This confirmed that this receptor is highly expressed in DA neurons also differentiated from this cell source, when transplanted to the striatum. Overlay of all three antigens confirmed the 5-HT6 receptor to reside in hESC-derived DA neurons (O). Scale bar, 100 µm (K) and 50 µm (N).

Fig 5: Comparison of VM- versus FB-Patterned Graft CompositionPhenotypic characterization of hESC-derived VM-patterned (A–F) and FB-patterned cells (G–L) 6 months after grafting to the nigra.(A, B, G, and H) VM- (A) and FB-patterned cells (G) generated grafts of comparable size, as visualized by human NCAM staining. VM- (B) but not FB-patterned grafts (H) generated TH+ neurons in vivo.(C and I) In VM-patterned grafts (C) but not FB-patterned grafts (I), TH+ neurons also co-expressed the midbrain DA neuron marker FOXA2 (arrowheads).(D–F and J–L) FB- (J) but not VM-patterned graft cells (HuNu+) (D) expressed NKX2.1 and FOXG1 (E and K), and included DARPP-32+ neurons (F and L). See also Figure S1 for in vitro characterization of cell preparations.All graft neurons expressed a rabies tracing construct and, thus, nuclear GFP. Scale bars represent 1 mm (A, B, G, and H) and 20 µm (C–F and I–L). Images in (A), (B), (G), and (H) are digitally stitched from multiple high-magnification images. DARPP-32, dopamine- and cAMP-regulated phosphoprotein; FB, forebrain; FOXA2, forkhead box A2; FOXG1, forkhead box protein G1; hESCs, human embryonic stem cells; HuNu, human nucleus; NKX2.1, NK2 homeobox 1; TH, tyrosine hydroxylase; Tx, transplant; VM, ventral midbrain.