Fig 2: Formation of 3D NE organoids from human iPSCs. A) Culture system for generating 3D DV patterned NE organoids from single hiPSCs. B) Developmental phases during the formation of NE organoids from single hiPSCs. Phase 1, hiPSCs were digested into single cells and seeded on Geltrex-coated glass coverslip on day 0. Phase 2, hiPSCs differentiated into NE cells and self-organized into cystic structures with smooth outer surface and containing central lumen with apical-basal polarity on day 6. Phase 3, development of NE cysts with FOXA2+ ventral NE cells and PAX3+ dorsal NE cells, forming DV patterned NE organoids. Top panels show bright-field images of hiPSCs, NE cysts, and NE organoids. Bottom panels show immunofluorescence staining of pluripotency marker SOX2, F-actin (phalloidin), N-cadherin, early neuroectodermal marker PAX6, dorsal marker PAX3, and ventral marker FOXA2 in corresponding phases during NE organoid formation. Scale bars: 100 µm. C) Representative bright-field (BF) images of NE cysts on day 7 and the corresponding immunofluorescence staining of PAX6, N-cadherin, and nuclei in NE cysts formed in the presence of 2%, 4%, and 8% Geltrex in the neural induction medium. Scale bars: 100 µm. D) Quantification of the number density, area, and lumen-to-cyst size ratio of NE cysts on day 7, n = 5. E) Representative bright-field (BF) images (scale bars: 100 µm) and the corresponding immunofluorescence staining (scale bars: 50 µm) of F-actin (phalloidin), PAX6, FOXA2, and nuclei of NE organoids on day 18 formed in the presence of 2%, 4%, and 8% Geltrex in the neural induction medium. F) Quantification of the number density, area, lumen-to-organoid size ratio, and the percentage of floor-plate patterned NE organoids on day 18, n = 3. Error bars in (D) and (F) represent S.E.M. P-values of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (not significant) P > 0.05. Using one-way analysis of variance (ANOVA) followed by a Tukey's multiple comparisons test.

Fig 3: Proposed model for the role of Ezh2 in mouse midbrain development. Ezh2 controls neural progenitor proliferation and brain area identity via direct and indirect mechanisms. Ezh2 regulates cell cycle progression by H3K27me3-mediated repression of negative cell cycle regulators Cdkna2 and Cdkn2c and Wnt/ß-catenin signaling inhibitors Wif1 and Dkk2. Also, Ezh2 maintains midbrain identity of cells by suppressing forebrain determinants Foxg1 and Pax6 and by maintaining Wnt/ß-catenin signaling, which is essential for the expression of midbrain markers Pax3 and Pax7. Therefore, loss of Ezh2 in the developing mouse midbrain leads to decreased proliferation and precocious cell cycle exit of neural progenitors in addition to a partial loss of midbrain identity and ectopic establishment of forebrain identity

Fig 4: Midbrain markers are not directly repressed by Pax6 but are regulated indirectly by Wnt/ß-catenin signaling. (a) A Pax6 overexpression construct together with a GFP-expressing vector (Pax6OE) or a GFP-expressing vector alone (Control) were delivered by in utero electroporation into the dorsal midbrain at E13.5. Two days later the proportion of GFP+ cells expressing midbrain markers Pax3 and Pax7 was analyzed. Overexpression of Pax6 does not affect the proportion of midbrain marker-expressing cells. n = 3 in each group, two different litters (also see Additional file 1: Figure S7B). (b, c) Immunostaining for Pax3 (b) and Pax7 (c) at E12.5 reveals decreased protein levels in midbrains with ablated Wnt/ß-catenin signaling (Ctnnb1 sign mt) compared to control. Cartoon insets show regular expression pattern, asterisk indicates basal. DAPI staining serves as nuclear marker: b, c; Scale bars: b, c, 500 µm (upper panel); b, c, 40 µm (lower panel); Error bars indicate SD; IUE, in utero electroporation; ctrl, control; Ctnnb1 sign mt, Wnt/ß-catenin signaling mutant; MB, midbrain

Fig 5: Photomicrograph of muscle sections in ischemic non-treated and ischemic treated groups by BM-MSCs stained for Pax3 and Pax7 nuclear expression. (a) The granulation tissue of the ischemic non-treated group showing negative expression of Pax3 (diaminobenzidine, original magnification, x200). (b) The granulation tissue of the ischemic non-treated group showing negative expression of Pax7 (diaminobenzidine, original magnification, x400). (c) The granulation tissue of the ischemic treated group by BM-MSCs showing cells with positive nuclear staining of Pax3 (diaminobenzidine, original magnification, x200). (d) The granulation tissue of the ischemic treated group by BM-MSCs showing cells with positive nuclear staining of Pax7 (diaminobenzidine, original magnification, x200)