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. 2025 Feb 11;31:101561. doi: 10.1016/j.mtbio.2025.101561

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© 2025 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Fig. 4 — Gel embedding yielded significantly large organoids and enhanced motor neuron differentiation efficiency.

a) Schematic illustration of the neuronal maturation protocol for SCOs. BDNF: Brain-derived neurotrophic factor; IGF: Insulin like growth factor; AA: Ascorbic acid; cAMP: Cyclic adenosine monophosphate.

b) Bright field images of SCOs at day 30. Scale bar = 1 cm.

c) Representative Immunofluorescence images of neural tube. Scale bars, 1 cm (top panel), 500 μm (bottom panel).

d) Immunofluorescence in SCOs cryo-sections at day 30 showing expression of motor neuron (ISL-1) marker. Scale bars, 500 μm (left panel), 100 μm (right panel).

e) Expansion area of the SCOs sphere at day 30(n = 8). Data are presented as mean ± SD.

f) Proportion of ISL-1⁺ nuclei in immunofluorescence staining images. Data are presented as mean ± SD.

g) Schematic diagram of the split-and-passage culture protocol for SCOs.

h) SCOs were passaged at day 20 by a 1:3 or 1:4 split with syringe needle.

i) Bright field images of P2-SCOs at day 30. Scale bar = 1mm.

j) Immunofluorescence staining of neural tube of P2-SCOs at D30. Scale bar = 100 μm.

k) Immunofluorescence staining of motor neurons (ISL-1) of P2-SCOs at D30. Scale bar = 100 μm.

l) Gene expression analysis of neuronal cell type markers in P2-SCOs at day 30 of in vitro differentiation. Data are presented as mean. Each cell represents two independent differentiations of a single cell line[Kruskal-Wallis test p < 0.0001(ESC9), p = 0.0001(iPSC2) for EVX1, p < 0.0001(ESC9), p = 0.0129(iPSC2) for EN1, p = 0.0004(ESC9), p = 0.0006(iPSC2) for CHX10, p < 0.0001(ESC9), p = 0.0006(iPSC2) for GATA3, p = 0.0003(ESC9), p = 0.0002(IPSC2) for HB9, p = 0.0351(ESC9), p = 0.0709(iPSC2) for ISL1, p = 0.3263(ESC9), p = 0.0045(iPSC2) for SIM1].

m) Gene expression analysis of neurotransmitter identity markers in P2-SCOs at day 30. Data are presented as mean. Each cell represents two independent differentiations of a single cell line[Kruskal-Wallis test p < 0.0001(ESC9), p < 0.0001(iPSC2) for CHAT, p = 0.0343(ESC9), p = 0.0012(iPSC2) for vGLUT2, p = 0.0010(ESC9), p = 0.5570(iPSC2) for GAD1, p = 0.2110(ESC9), p = 0.0491(iPSC2) for GLYT2].