Characterization of Hypothalamic MCH Neuron Development in a 3D Differentiation System of Mouse Embryonic Stem Cells

Generation of MCH neurons in ES-Hypo. A, The qRT-PCR-based analysis of the Pmch expression during ES-Hypo differentiation. Pmch encodes the precursor of MCH. Data were normalized to Actb and gene expression on day 14 and plotted in log10 scale. n = 3 experiments. **p < 0.01 versus day 14 by Dunnett test. B, Immunofluorescence staining of ES-Hypo for MCH on days 15, 22, and 36. Nuclei were stained with DAPI. The right panels show high-magnification images of the boxed regions in the middle panels. Scale bars: 50 μm (low magnification) and 10 μm (high magnification). C, Quantification of the cell body diameter for MCH-ir cells on days 22 and 36. The long and short diameters for each MCH-ir cell were measured as shown in the right panel. n = 300 cells per day. ****p < 0.0001 by Welch’s t test. D–F, The analysis of MCH-ir cells in the dissociation culture. Cells were dissociated from ES-Hypo on days 19–20 or 30–33 and cultured in a monolayer for 3 d. Representative morphologies of MCH-ir cells are shown in fluorescence images (D) and a cell trace (F). Scale bars: 20 μm (D) and 50 μm (F). The percentage of MCH-ir cells showing unipolar, bipolar, or multipolar morphology is presented in the pie charts (E) for days 22–23 (n = 272 cells) and days 33–36 (n = 200 cells). Functional properties of MCH-ir cells on day 36 were evaluated by calcium imaging as shown in Extended Data Figure 2-1. G, Representative images of day-36 mESC aggregates immunostained for MCH and HuC/D. The aggregates were initially differentiated in gfCDM (left, corresponding to ES-Hypo) or gfCDM + KSR (right). Arrowheads indicate double-positive cells. Scale bar: 20 μm. H, The percentage of MCH⁺HuC/D⁺ cells among total HuC/D⁺ cells under the culture conditions shown in G. n = 8 aggregates per condition. ****p < 0.0001 by Welch’s t test.

Reciprocal connectivity between MCH and orexin neurons in ES-Hypo. A, Representative images of ES-Hypo immunostaind for orexin on days 24 and 32. The inset shows a magnified view of an orexin-ir cell in the boxed region. Scale bars: 50 and 10 μm (inset). B, The qRT-PCR-based analysis of the Hcrt expression on days 14 and 31. Hcrt encodes the precursor of orexin. Data were normalized to Actb and gene expression on day 14 and plotted in log10 scale. n = 3 experiments. *p < 0.05 by Welch’s t test. C–E, Double-immunofluorescence images showing putative connections between MCH-ir and orexin-ir cells in ES-Hypo on days 32 and 36. The boxed region in C is magnified in D. Two orexin-ir cells are contacted by MCH-ir fibers (D, arrows) or boutons (D, arrowheads). Similarly, an MCH-ir cell is closely apposed by orexin-ir boutons (E, arrowheads). Scale bars: 10 μm.

Characterization of neuropeptide/neurotransmitter phenotype of MCH neurons in ES-Hypo. A, Representative images of day-36 ES-Hypo immunostained for MCH and different neurochemical markers GAD67, VGLUT2, nesfatin-1, and CART. Scale bars: 10 μm. B, The percentage of MCH⁺ cells expressing different neurochemical markers. n = 3–4 aggregates per marker.

The generation of CART-negative MCH neurons and orexin neurons is increased by the activation of Hh signaling in ES-Hypo. A, The regional expression of transcription factors in the telencephalon and anterior diencephalon of embryonic mouse brain around E12. The indicated expression patterns are based on the published literature (Shimamura et al., 1995; Marín et al., 2002; Shimogori et al., 2010; Lu et al., 2013; Díaz et al., 2014; Ferran et al., 2015). Cx, cortex; MGE, medial ganglionic eminence; OS, optic stalk; PTh, prethalamus; zli, zona limitans intrathalamica. B, Representative images of day-7 mESC aggregates immunostained for Rax and Pax6 (upper panels) or Nkx2.1 (lower panels). The aggregates were differentiated in the absence (−) or presence (+) of 30 nm SAG. Scale bar: 100 μm. Immunostaining for the general neural progenitor marker Sox1 is presented in Extended Data Figure 5-1. C, The percentage of Rax⁺ cells expressing Pax6 (top) or Nkx2.1 (bottom) on day 7 under SAG (−) and (+) conditions. n = 10 aggregates per condition. ****p < 0.0001 by Welch’s t test. D, Serial sections from a day-7 aggregate cultured with SAG. The sections were stained for Foxg1 (#1), Rax (#2), or Nkx2.1/Nkx2.2 (#3). A Rax⁻ region is surrounded by dashed lines. Scale bar: 100 μm. E, Representative immunofluorescence images of MCH⁺CART⁺ (left) and MCH⁺CART⁻ (right) cell clusters in SAG-treated aggregates on day 30. In the MCH⁺CART⁻ cluster, only one MCH⁺ cell is weakly stained for CART (arrow). Scale bar: 20 μm. In Extended Data Figure 5-2, we assessed the co-expression of MCH and HuC/D in SAG-treated aggregates. In Extended Data Figure 5-3, we assessed the co-expression of CART and NK3R in SAG-treated aggregates. F, The percentage of MCH⁺CART⁻ cells among total MCH⁺ cells on day 30 under SAG (−) and (+) conditions. n = 8 aggregates per condition. **p < 0.01 by Welch’s t test. G, Representative images of SAG-treated (right) and untreated (left) aggregates immunostained for orexin on day 30. Arrows indicate orexin-ir cells. The inset shows a magnified view of an orexin-ir cell in the boxed region. Scale bars: 100 and 20 μm (inset). H, Quantification of orexin⁺ cells on day 30 under SAG (−) and (+) conditions. n = 12 aggregates per condition. ****p < 0.0001 by Brunner–Munzel test. I, Representative images of an MCH⁺CART⁺ (top) or MCH⁺CART⁻ (bottom) cell, which is contacted by orexin-ir boutons (arrowheads). Triple immunostaining was performed in SAG-treated aggregates on day 36. Scale bar: 10 μm.

Characterization of neuronal differentiation in SAG-treated ES-Hypo. A–G, Serial sections from SFEBq-cultured mESC aggregates (with SAG) on days 7 and 13. The sections were immunostained for Nkx2.1/Nkx2.2 (#1), Nkx2.1/HuC/D (#2), or Nkx2.1/Sox1 (#3). The day-13 aggregate contains a HuC/D⁺ neuron-dense area (B–D) and a Sox1⁺ rosette structure (E–G). Scale bars: 100 μm (A) and 50 μm (B–G). H, Representative images of SAG-treated mESC aggregates immunostained for MCH/Nkx2.1 (top) and MCH/Nkx2.2 (bottom) on day 22. Arrows indicate double-positive cells. Scale bar: 20 μm. I, The percentage of MCH-ir cells expressing Nkx2.1 or Nkx2.2 on day 22. n = 4–5 aggregates per marker. J, Triple immunostaing of SAG-treated mESC aggregates for MCH/CART/Nkx2.2 on day 30. Representative images of MCH⁺CART⁺ (left) and MCH⁺CART⁻ (right) cell clusters are shown, and MCH⁺Nkx2.2⁺ cells are indicated by arrows. Scale bar: 20 μm.

Characterization of neuronal differentiation in SAG-free ES-Hypo. A, FACS sorting of Rax::GFP⁺ and GFP⁻ cells from SFEBq-cultured mESC aggregates (without SAG) on day 7. B, Immunofluorescence images of Rax::GFP⁺ (right) and GFP⁻ (left) cell aggregates. FACS-sorted GFP⁺ and GFP⁻ cells were reaggregated and cultured until day 30 before staining for MCH. Nuclei were stained with DAPI. Scale bar: 50 μm. C, Serial sections from a day-7 aggregate cultured without SAG. The sections were immunostained for GFP/Sox1/Nkx2.2 (#1), GFP/Pax6/Nkx2.1 (#2), or GFP/Foxg1 (#3). Scale bar: 100 μm. D–J, Immunofluorescence analysis of GFP⁻ cell aggregates on days 13 and 22. Two serial sections from a day-13 aggregate were stained for GFP/Pax6 or Nkx2.1/Sox1/HuC/D (D–F). Two sections from a day-13 aggregate were stained for Nkx2.1/Nkx2.2 or Sox1/Nkx2.2 (G–I). Day-22 aggregates were stained for MCH/Nkx2.1 or MCH/Nkx2.2 (J, arrows indicate double-positive cells). Scale bars: 100 μm (D, G), 20 μm (E, F, H, I), and 10 μm (J). K, The percentage of MCH-ir cells expressing Nkx2.1 or Nkx2.2 in GFP⁻ cell aggregates on day 22. n = 4 aggregates per marker.