These experiments were approved by the ethics committee of Nagoya University and performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.
Generation of FNDI hiPSCs and a control cell line
We generated iPSCs from a patient with FNDI with a Cys98Stop mutation3 based on ethical approval from Nagoya University Committee (2013-0228-2). To establish patient-derived iPSCs, peripheral blood monocular cells of the patient were reprogrammed using episomal vectors (expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, EBNA1, and p53shRNA) as described previously59,60. The iPSCs showed embryonic stem cell-like morphology and normal karyotype (Fig. S3A). These cells expressed the undifferentiated markers (Fig. S3B, C) and could differentiate into the three germ layers in vitro (Fig. S3D), indicating that these cells were pluripotent (deposited in RIKEN BRC as HPS1011 and HPS1904). We named them as FDI-02. As a control, we used the 201B7 cell line [Research Resource Identifier (RRID): CVCL_A324].
Thawing and maintenance of primed hiPSCs
We prepared mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C treatment on 0.1% gelatin-coated dishes (1.2 ×106 cells/10 cm dish). Primed hiPSCs frozen in liquid nitrogen were thawed as quickly as possible in warmed maintenance medium, comprising DMEM/F12 (D6421; Sigma, St. Louis, Missouri, USA) supplemented with 20% (v/v) KSR (lot No. 1517496; Invitrogen, Waltham, Massachusetts, USA), 0.1 mM non-essential amino acids (11140-050; Gibco, Waltham, MA, USA), 2 mM L-glutamine (25030-081; Gibco), 5 ng/mL recombinant human basic FGF (068-04544; Wako, Osaka, Japan), and 0.1 mM 2-mercaptoethanol (131-14572; Wako. Cells suspended in maintenance medium were distributed on MEF-coated dishes and maintained in a CO2 incubator under 2% CO2 at 37°C. The medium was changed daily.
For passaging, primed hiPSC colonies were harvested by incubation in 0.25% (w/v) trypsin and 0.1 mg/mL collagenase IV in PBS containing 20% (v/v) KSR and 1 mM CaCl2 for 6–8 min at 37°C. The harvested clumps were broken into smaller pieces by gentle pipetting. The passages were performed at a split ratio of 1:4–6.
Reset of hiPSCs from the primed state to naive state and maintenance incubation
hiPSCs harvested during passaging, were suspended in mTeSR1TM medium (ST-85850; STEMCELL Technologies, Vancouver, Canada), distributed on a Matrigel-coated dish (354277; Corning, Corning, New York, USA), and incubated under 5% CO2 at 37°C. After 24–36 h, the mTeSR1 medium was replaced with RSeTTM feeder-free medium (ST-05975; STEMCELL Technologies) and cells were incubated under hypoxic conditions (5% O2, 5% CO2) at 37°C. The medium was changed every second day. Reset to the naive state was confirmed by changes in colony morphology and immunohistochemistry.
For passaging, naive hiPSCs were harvested using TrypLE Express (12605-010; Thermo Fisher, Waltham, Massachusetts, USA). Cells were counted and 1.1 × 106 cells were distributed on a 10 cm Matrigel-coated dish. The medium was changed after 24–36 h and thereafter, every second day. This passaging procedure was performed every 4–6 days.
Thawing and maintenance of primed hiPSCs
Hypothalamic differentiation was performed using the SFEBq culture. Primed hiPSCs were harvested and dissociated into single cells using TrypLE Express (12605-010; Invitrogen) containing 0.05 mg/mL DNase I (11284932001; Roche, Basel, Switzerland) and 10 mM Y-27632 (034-24024; Wako). Cells were then suspended in gfCDM differentiation medium [1:1 Iscove's-modified Dulbecco's medium (IMDM), GlutaMAXTM supplement (31980-030; Gibco)/Ham's F-12 nutrient mix, GlutaMAX™ supplement (31765-035; Gibco), 250 mg/mL BSA (A3156; Sigma), 1× chemically defined lipid concentrate (11905-031; Gibco), and 438 µM 1-thioglycerol (M6145; Sigma)] and distributed in low-cell-adhesion 96-well plates with V-bottomed conical wells (MS-9096V; Sumitomo Bakelite, Tokyo, Japan) at 5,000 cells in 100 µL/well. When the SFEBq culture was started, gfCDM contained 0.7–5.0% KSR and 20 mM Y-27632, which were diluted in a step-wise manner by replacing one-half of the medium with new medium without KSR or Y-27632. In another method, harvested primed hiPSCs were broken by gentle pipetting. Eighty percent of the broken clumps from one confluent 10 cm dish were suspended in gfCDM and distributed into one 96-well plate. Naive hiPSCs were harvested using TrypLE Express and dissociated into single cells without mechanical disruption. These were then suspended in gfCDM (5,000 cells in 100 µL/well) and distributed in 96-well plates. All SFEBq cultures were incubated in 5% CO2 at 37°C. The concentrations of KSR and Y-27632 in the medium used for the medium change were adjusted for each experiment.
We designated the day when the culture was started as day 0. On day 3, 100 µL gfCDM was added to each well. From day 6, half of the medium was changed every 3 days. After culturing in a 96-well plate for 30 days, the aggregates were transferred to a 10 cm dish for suspension culture. From day 60, 100 ng/mL FGF8b (423-F8; R&D Systems, Minneapolis, MN, USA) and 5 µg/mL heparin (NIPRO, Osaka, Japan) were added to the differentiation medium. From day 90, the medium was completely replaced with DMEM/F12 supplemented with glucose, N2, and B27 [DFNB medium: DMEM/F12 (D8900; Sigma) supplemented with 3.85 g/L glucose (07-0680-5; Sigma), 1.2 g/L sodium hydrogen carbonate (28-1850-5; Sigma), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) (15140-122; Gibco), N2 (175020-01; Gibco), B27 (125870-01; Gibco), and 10 ng/mL CNTF (257-NT; R&D Systems)] with 100 ng/mL FGF8b and 5 µg/mL heparin.
Immunohistochemistry
Aggregates were fixed with 4% PFA for 10–15 min and embedded in OCT compound (4583; Sakura Finetek, Tokyo, Japan). Ten micron thick sections were cut using a cryostat, mounted on slides, and fixed in 4% PFA for 10 min.
Sections were permeabilized using 0.3% Triton X-100/PBS and washed with PBS. Subsequently, they were incubated in 2% (w/v) skimmed milk/PBS for 1 h at RT and then with primary antibodies diluted in 2% skimmed milk/PBS, overnight at 4°C. The next day, they were washed with 0.05% Tween 20/PBS and reacted with 4,6-diamidino-2-phenylindole (DAPI; D523; Dojindo, Kumamoto, Japan) and secondary antibodies diluted in 2% skimmed milk/PBS for 2 h at RT. Subsequently, they were washed with 0.05% Tween 20/PBS and mounted in SlowFade™ Diamond (S36972; Thermo Fisher Scientific).
Primary antibodies were used against the following molecules (with dilutions): AVP (T5048; guinea pig; 1:2000; Peninsula; RRID:AB_2313978), BRN2 (sc-6029; goat; 1:500; Santa Cruz; RRID:AB_2167385), Copeptin (BORIS Y; rabbit; 1:1000; Woomera Therapeutics), E-cad (M108; rat; 1:50; TAKARA), FOXG1 (M227; rabbit; 1:1000; TAKARA; RRID:AB_2827749), mutant NPII [AFT965002-B(2B); rabbit; 1:1000; custom], NANOG (4903; rabbit; 1:500; Cell signaling; RRID:AB_10559205), NKX2.1 (16108; mouse; 1:100; PROGEN; RRID:AB_1543129), NPII (MABN845; mouse; 1:1000; Millipore; RRID:AB_2819363), OCT3/4 (611202; mouse; 1:100; BD biosciences; RRID:AB_398736), OTP (MS1535GS; guinea pig; 1:1000; Takara), PAX6 (ab195045; rabbit; 1:350; abcam; RRID:AB_2750924), SMA (M0851; mouse; 1:500; DAKO; RRID:AB_2223500), SOX17 (81778; rabbit; 1:3000; Cell Signaling; RRID:AB_2650582), SSEA-4 (MAB4304; mouse; 1:2000; Millipore; RRID:AB_177629), TFCP2L1 (AF5726; goat; 1:200; R&D Systems; RRID:AB_2202564), TRA1-60 (MAB4360; mouse; 1:200; Millipore; RRID:AB_2119183), and TUJ1 (MMS-435P; mouse; 1:500; Covance; RRID:AB_2313773).
Statistics and reproducibility
We have described the exact n values for each experiment in the main text and figure legends. IBM SPSS Statistics (IBM, Armonk, New York, USA) was used for the statistical analyses. Two-group comparisons were performed using the two-tailed unpaired t-test. Significance was set at P < 0.05.