REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse Monoclonal Anti-Beta III Tubulin (Clone 2G10)	Abcam	Cat#ab78078; RRID: AB_2256751
Rabbit Polyclonal Anti-GAPDH	Abcam	Cat#Ab9485; RRID: AB_307275
Mouse Monoclonal Anti-Glial Fibrillary Acidic Protein (Clone GA5)	Millipore	Cat#MAB360; RRID: AB_11212597
Rabbit Polyclonal Anti-OTP	Thermo Fisher Scientific	Cat#PA5-31513; RRID: AB_2548986
Rabbit Monoclonal Anti-POMC (EPR17571)	Abcam	Cat#Ab210605; RRID: N/A
Rabbit Polyclonal Anti-RFP	Rockland	Cat#600-401-379; RRID: AB_2209751
Rabbit Monoclonal Anti-SOX9 (EPR14335-78)	Abcam	Cat#Ab185966; RRID: AB_2728660
Mouse Monoclonal Anti-TH (Clone LNC1)	Millipore	Cat#MAB318; RRID: AB_2201528
Rabbit Polyclonal Anti-TRH	Thermo Fisher Scientific	Cat#PA5-97006; RRID: AB_2808808
Rabbit Monoclonal Anti-TTF1 (NKX2-1) (Clone EPR5955(2))	Abcam	Cat#Ab133737; RRID: AB_2811263
Mouse Monoclonal Anti-VGLUT2 (Clone CL2921)	Atlas Antibodies	Cat#AMAb91081; RRID: AB_2665793
Biological samples
Postmortem Hypothalamus (PCR)	Neuropathology Brain Bank and Research CoRE, Mount Sinai	Cat#NPBB023; RRID: N/A
Postmortem Hypothalamus (IHC)	Neuropathology Brain Bank and Research CoRE, Mount Sinai	Cat#NPBB127; RRID: N/A
Chemicals, peptides, and recombinant proteins
Accutase Cell Detachment Solution	Innovative Cell Technologies	Cat#AT104; RRID: AB_2869384
B-27™ Supplement (50X), Minus Vitamin A	Gibco	Cat#12587010; RRID: N/A
Blotto, Non-Fat Dry Milk	ChemCruz	Cat#sc-2324; RRID: N/A
Bovine Albumin Fraction V (7.5% solution)	Gibco	Cat#15260037; RRID: N/A
DAPT	Cayman Chemical	Cat#13197; RRID: N/A
DB-cAMP / Dibutyryl-cAMP	BioLog	Cat#D 009; RRID: N/A
DMEM/F-12, GlutaMAX™ Supplement	Gibco	Cat#10565018; RRID: N/A
DNase Vial (D2)	Worthington Biochemical Corporation	Cat#LK003170; RRID: N/A
Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix	Gibco	Cat#A1413302; RRID: N/A
HBSS with 10 mM HEPES, Without Phenol Red	STEMCELL Technologies	Cat#37150; RRID: N/A
Hibernate A	BrainBits	Cat#HA; RRID: N/A
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water	Thermo Fisher Scientific	Cat#H3570; RRID: N/A
Laminin Mouse Protein, Natural	Gibco	Cat#23017015; RRID: N/A
L-Ascorbic Acid (White Crystalline Powder)	Fisher Scientific	Cat#BP351-500; RRID: N/A
LDN 193189 Dihydrochloride	Tocris	Cat#6053; RRID: N/A
Leibovitz's L-15 Medium	Gibco	Cat#11415064; RRID: N/A
N-2 Supplement (100X)	Gibco	Cat#17502048; RRID: N/A
Normal Goat Serum	Thermo Fisher Scientific	Cat#31872; RRID: N/A
PBS, pH 7.4 (-CaCl2, -MgCl2)	Gibco	Cat#10010023; RRID: N/A
PDS Kit, Papain Vial	Worthington Biochemical Corporation	Cat#LK003176; RRID: N/A
Penicillin-Streptomycin (10,000 U/mL)	Gibco	Cat#15140122; RRID: N/A
Pierce™ 16% Formaldehyde (w/v), Methanol-free	Thermo Fisher Scientific	Cat#28908; RRID: N/A
Poly-L-Ornithine Hydrobromide	Sigma-Aldrich	Cat#P3655; RRID: N/A
Ponceau S Solution	Sigma-Aldrich	Cat#P7170; RRID: N/A
Purmorphamine	STEMCELL Technologies	Cat#72204; RRID: N/A
Recombinant Human BDNF Protein	R&D Systems	Cat#248-BD; RRID: N/A
Recombinant Human GDNF Protein	R&D Systems	Cat#212-GD; RRID: N/A
SAG	Cayman Chemical	Cat#11914; RRID: N/A
SB431542	Stemgent	Cat#04-0010-10; RRID: N/A
StemFlex™ Medium	Gibco	Cat#A3349401; RRID: N/A
Thiazovivin	Selleck Chemicals	Cat#S1459; RRID: N/A
Triton™ X-100 Surfact-Amps™ Detergent Solution	Thermo Fisher Scientific	Cat#85111; RRID: N/A
XAV939	Tocris	Cat#3748; RRID: N/A
Y-27632 Dihydrochloride	Tocris	Cat#1254; RRID: N/A
Critical commercial assays
4X Bolt™ LDS Sample Buffer	Thermo Fisher Scientific	Cat#B0007; RRID: N/A
Antibody Dilution Buffer	Roche Diagnostics	Cat#05280524001; RRID: N/A
cOmplete™, Mini Protease Inhibitor Cocktail	MilliporeSigma	Cat#11836153001; RRID: N/A
High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems	Cat#4368814; RRID: N/A
Maestro Multiwell 768 channel MEA system with USB	Axion Biosystems	Cat# AAVMAESTRO768DEA; RRID: N/A
Monarch® Total RNA Miniprep Kit	New England BioLabs	Cat#T2010S; RRID: N/A
NuPAGE™ MOPS SDS Running Buffer (20X)	Thermo Fisher Scientific	Cat#NP0001; RRID: N/A
Phosphatase Inhibitor Cocktail II	Abcam	Cat#ab201113; RRID: N/A
Pierce™ BCA Protein Assay Kit	Thermo Fisher Scientific	Cat#23225; RRID: N/A
PowerUp SYBR Green Master Mix	Applied Biosystems	Cat#A25742; RRID: N/A
Q5® Hot Start High-Fidelity 2X Master Mix	New England BioLabs	Cat#M0494S; RRID: N/A
RIPA Lysis and Extraction Buffer	Thermo Fisher Scientific	Cat#89900; RRID: N/A
TBS Buffer, 20X Liquid	VWR	Cat#10791-796; RRID: N/A
TWEEN® 20	Sigma-Aldrich	Cat#P1379; RRID: N/A
ULTRA Cell Conditioning (ULTRA CC1)	Roche Diagnostics	Cat#950-224; RRID: N/A
Western Lightning Plus, Chemiluminescent Substrate (130 mL)	PerkinElmer	Cat#NEL103001EA; RRID: N/A
Deposited data
scRNA-seq Dataset (GEO)	This paper	GSE237274
scRNA-seq Dataset - processed (Mendeley Data)	This paper	https://doi.org/10.17632/nxtkn2w28v.1
Customs Codes and Script for scRNA-seq Dataset (GitHub)	This paper	https://github.com/powellgenomicslab/hypothalamus_organoid_scRNA
Experimental models: Cell lines
Human: H1 (WA01) hESCs	WiCell Research Institute	Cat#WA01; RRID: CVCL_9771
Human: KOLF2.1J hiPSCs	The Jackson Laboratory	Cat#KOLF2.1J; RRID: N/A
Oligonucleotides
Primer: ACTB Forward: CCTGGCACCCAGCACAAT	Pilegaard et al.72	RRID: N/A
Primer: ACTB Reverse: GCCGATCCACACGGAGTACT	Pilegaard et al.72	RRID: N/A
Primer: DLX5 Forward: GTCTTCAGCTACCGATTCTGAC	PrimerBank	Cat#84043959c3; RRID: N/A
Primer: DLX5 Reverse: CTTTGCCATAGGAAGCCGAG	PrimerBank	Cat#84043959c3; RRID: N/A
Primer: FOXG1 Forward: TGGCCCATGTCGCCCTTCCT	Nolbrant et al.73	RRID: N/A
Primer: FOXG1 Reverse: GCCGACGTGGTGCCGTTGTA	Nolbrant et al.73	RRID: N/A
Primer: GAD2 Forward: TTTTGGTCTTTCGGGTCGGAA	PrimerBank	Cat#197276619c1; RRID: N/A
Primer: GAD2 Reverse: TTCTCGGCGTCTCCGTAGAG	PrimerBank	Cat#197276619c1; RRID: N/A
Primer: GFAP Forward: CTGCGGCTCGATCAACTCA	PrimerBank	Cat#334688843c1; RRID: N/A
Primer: GFAP Reverse: TCCAGCGACTCAATCTTCCTC	PrimerBank	Cat#334688843c1; RRID: N/A
Primer: HCRT Forward: TTCCACAAAGGTCTCCTGGG	This paper	RRID: N/A
Primer: HCRT Reverse: TAGAGGCGGCAAGAGCAAG	This paper	RRID: N/A
Primer: NKX2-1 Forward: TGAGTCCAAAGCACACGACTCC	This paper	RRID: N/A
Primer: NKX2-1 Reverse: CCTCCATGCCCACTTTCTTGTA	This paper	RRID: N/A
Primer: NR5A1 Forward: GGGTGGCCGGAACAAGTTT	PrimerBank	Cat#169881245c2; RRID: N/A
Primer: NR5A1 Reverse: GCCCGAATCTGTGCCTTCT	PrimerBank	Cat#169881245c2; RRID: N/A
Primer: OTP Forward: CGACATCTTTATGCGTGAGGAG	This paper	RRID: N/A
Primer: OTP Reverse: GTCTTTTTGCGCTTCTTCCACT	This paper	RRID: N/A
Primer: POMC Forward: CTCCCGAGACAGAGCCTCA	Flori et al.74	RRID: N/A
Primer: POMC Reverse: CACTCCAGCAGGTTGCTTTC	This paper	RRID: N/A
Primer: RAX Forward: AAGCCCCTCGACCCTACTG	PrimerBank	Cat#126116580c2; RRID: N/A
Primer: RAX Reverse: CCGCCGATGCTTTTTCTTGG	PrimerBank	Cat#126116580c2; RRID: N/A
Primer: SLC17A6 Forward: TGGACATGGTCAACAACAGCA	PrimerBank	Cat#215820654c2; RRID: N/A
Primer: SLC17A6 Reverse: GGAACCGTGGATCATCCCC	PrimerBank	Cat#215820654c2; RRID: N/A
Primer: SOX9 Forward: GTACCCGCACTTGCACAAC	Masuda et al.75	RRID: N/A
Primer: SOX9 Reverse: TCTCGCTCTCGTTCAGAAGTC	Masuda et al.75	RRID: N/A
Primer: SST Forward: ACCCAACCAGACGGAGAATGA	PrimerBank	Cat#71979669c1; RRID: N/A
Primer: SST Reverse: GCCGGGTTTGAGTTAGCAGA	PrimerBank	Cat#71979669c1; RRID: N/A
Primer: TAC1 Forward: CAGCGACCAGATCAAGGAGG	This paper	RRID: N/A
Primer: TAC1 Reverse: GGGCGATTCTCTGCAGAAGA	This paper	RRID: N/A
Primer: TH Forward: GGAAGGCCGTGCTAAACCT	PrimerBank	Cat#88900504c1; RRID: N/A
Primer: TH Reverse: GGATTTTGGCTTCAAACGTCTC	PrimerBank	Cat#88900504c1; RRID: N/A
Primer: TRH Forward: TCCTCCGGGAAAACATCCAG	PrimerBank	Cat#306518589c1; RRID: N/A
Primer: TRH Reverse: GGAGAGCCAGTCAGATTGAAAGA	PrimerBank	Cat#306518589c1; RRID: N/A
Software and algorithms
Aperio ImageScope	https://www.leicabiosystems.com/digital-pathology/manage/aperio-imagescope/	RRID: SCR_020993
AxIS	https://www.axionbiosystems.com/products/axis-software	RRID: SCR_016308
CellProfiler Image Analysis Software	https://cellprofiler.org/	RRID: SCR_007358
Cell Ranger	https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger	RRID: SCR_017344
Cyclone	Scialdone et al.76	RRID: N/A
Demuxafy	Neavin et al.77	RRID: N/A
DropletQC	Muskovic and Powell78	RRID: N/A
FSC Express 7	https://denovosoftware.com/	RRID: N/A
Geneious Prime	http://www.geneious.com/	RRID: SCR_010519
GraphPad Prism	http://www.graphpad.com/	RRID: SCR_002798
MAST	Finak et al.79	RRID: SCR_016340
Microsoft Excel	https://www.microsoft.com/en-gb/	RRID: SCR_016137
R	https://www.r-project.org/	RRID: SCR_001905
scDblFinder	Germain et al.80	RRID: SCR_022700
scds (Single Cell Doublet Scoring)	Bais and Kostka81	RRID: SCR_021541
scPred	Alquicira-Hernandez et al.29	RRID: N/A
scVelo	https://github.com/theislab/scvelo	RRID: SCR_018168
Seurat	https://satijalab.org/seurat/	RRID: SCR_007322
Velocyto	http://velocyto.org/	RRID: SCR_018167
Other
Aperio VERSA Brightfield, Fluorescence & FISH Digital Pathology Scanner	Leica Biosystems	RRID: N/A
BD FACSAria II Cell Sorter	BD Biosciences	RRID: SCR_018934
Benchmark Special Stains Stainer Module	Roche Diagnostics	Cat#06468373001; RRID: N/A
CellInsight CX7 HCS Platform	Thermo Fisher Scientific	RRID: N/A
Corning® 125 mL Disposable Spinner Flask with 70 mm Top Cap and 2 Angled Sidearms, Sterile	Corning	Cat#3152; RRID: N/A
CytoView MEA Plate 48	Axion Biosystems	Cat#M768-tMEA-48B; RRID: N/A
Eclipse Ts2R-fl	Nikon	RRID: N/A
Falcon® Round-Bottom Tubes with Cell Strainer Cap, 5 mL	STEMCELL Technologies	Cat#100-0087; RRID: N/A
Fisherbrand™ Sterile Cell Strainers (70 μm)	Fisher Scientific	Cat#22-363-548; RRID: N/A
iBlot™ 2 Gel Transfer Device	Thermo Fisher Scientific	Cat#IB21001; RRID: N/A
iBlot™ Transfer Stack, PVDF, mini	Thermo Fisher Scientific	Cat#IB401002; RRID: N/A
NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels	Thermo Fisher Scientific	Cat#NP0321BOX; RRID: N/A
NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels	Thermo Fisher Scientific	Cat#NP0323BOX; RRID: N/A
pluriStrainer® 300 μm, 25 pcs. – Sterile (Cell Strainer)	pluriSelect	Cat#43-50300-03; RRID: N/A
QuantSudio 7 Flex Real-Time PCR System	Applied Biosystems	Cat#4485701; RRID: N/A
Stirrers, Magnetic, Nine-position, Dura-Mag	Chemglass Life Sciences	Cat#CLS-4100-09; RRID: N/A