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. 2018 Feb 26;8:3615. doi: 10.1038/s41598-018-22053-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of AVP-secreting hypothalamic neurons. (A) Immunostaining of a day 80 aggregate containing OTP+ cells. Few AVP+ cells were found in Transwell-cultured aggregates. AVP (green), OTP (red). (B) Schematic of 2D dissociation culture. Dissociated single cells from aggregates were attached onto coated glass-slips. (C) Effect of Akt-inhibitor on AVP induction. Immunostaining of day 150 aggregates with (right panel) or without (left panel) Akt-inhibitor treatment. AVP (red). (D) Schematic of the culture procedure for dorsal hypothalamic differentiation. (E) Number of AVP+ neurons with/without Akt-inhibitor treatment. n = 3. (F–M) Immunostaining for hypothalamic neurons in dissociation cultures treated with Akt-inhibitor. OXT (F, red), CRH (G, red), TRH (H, red), NPY (I, red), MCH (J, red), OREXIN (K, red), AGRP (L, red) and POMC (M, red). (N) MCH (red) immunostaining of an aggregate from ventral hypothalamus inducing conditions. (O) RAX and PAX6 positive rate with or without Akt-inhibitor treatment. Concentration of BMP4, SAG, and Akt-inhibitor was 1.5 nM, 0.5 μM, and 0.5 μM, respectively. RAX+/PAX6+ indicates double RAX/PAX6-positive cells. n = 3. (P) RAX positive rate with 0.5 μM SAG, 0.5 μM Akt-inhibitor, and 0.5-2.0 nM BMP4. n = 3. (Q) Number of immunopositive neurons per 1000 nuclei. n = 3. (R) AVP concentrations measured by radioimmunoassay in conditioned media of day 150 cells cultured with the Fig. 4D protocol. (C) control, using hESC-derived neocortex cells³⁸. (H) hypothalamus, using dorsal hypothalamic neurons. n = 10. (S) AVP-release analysis measured by radioimmunoassay with or without high K stimulation on day 150. ACSF: artificial cerebrospinal fluid. n = 10. (T) pro-MCH analysis by quantitative PCR with or without nerve growth factor (NGF), dexamethasone (DEX) and lithium chloride (LiCl) stimulation on day 150.C: control, using non-hypothalamic differentiated aggregates. H: hypothalamus, using aggregates of ventral hypothalamic differentiation. (−): no stimulation. (+): with stimulation of NGF, DEX and LiCl. n = 6. For all relevant panels, nuclear counter staining: DAPI (blue), scale bar: 50 μM. Error bars represent s.e.m. *P < 0.05, **P < 0.01.