Journal: Military Medical Research

Article Title: Engineering of functional auditory neurons from human induced pluripotent stem cells

doi: 10.1016/j.mmr.2026.100008

Figure Lengend Snippet: Stepwise differentiation of hiPSCs into human ONPs. a Overview and timeline of human SGN-like neuron differentiation protocol from hiPSCs and matching bright-field images. Scale bar=100 μm. b Comparison of cells at D25 and D63 following seeding at different cell densities (25,000 or 60,000 cells/cm 2 ) on D11 after sorting with NGFR beads. Scale bar=100 μm. c Immunostaining for NGFR + NESTIN + precursors of PPE and neural crest cells at D12. Scale bar=100 μm. d - f Immunostained D25 cells. Antibodies highlight neuroblast cells (NEUROD1 + ) and otic lineage cells (PAX2 + , PAX8 + , and SOX2 + ), but no expression of neuronal markers (TUBB3) at this stage. Scale bar=100 μm. g Bar graphs showing the average proportion (%) of positive cells on D25 cells stained with NEUROD1, PAX2, PAX8, SOX2, and TUBB3. Error bars represent SD. hiPSCs. Human induced pluripotent stem cells; ONPs. Otic neural progenitors; BMP4. Bone morphogenetic protein 4; LDN. LDN-193189; IWP2. Inhibitor of Wnt production-2; FGF2. Basic fibroblast growth factor 2; NGFR. Nerve growth factor receptor; Wnt3. Wnt family member 3; IGF-1. Insulin-like growth factor 1; SHH. Sonic hedgehog; RA. Retinoic acid; EGF. Epidermal growth factor; NEUROD1. Neurogenic differentiation 1; cAMP. Cyclic adenosine monophosphate; NT3. Neurotrophin 3; BDNF. Brain-derived neurotrophic factor; TUBB3. Tubulin beta 3 class III; PRPH. Peripheral neuronal marker peripherin; MPZ. Myelin protein zero; PPE. Pre-placodal ectoderm; SGC. Satellite glial cells; D. Day; SD. Standard deviation; GFAP. Glial fibrillary acidic protein; PAX2. Paired box gene 2; SOX2. SRY-box transcription factor 2; DAPI. 4′;6-diamidino-2-phenylindole.

Article Snippet: On D3, the medium was replaced with a CDM containing 10 ng/ml bone morphogenetic protein 4 (BMP4; Cat# 314-BP, R&D Systems, USA), 1 μmol/L SB-431542 (Cat# 1614, TOCRIS, UK), and 10 ng/ml basic fibroblast growth factor 2 (FGF2, Cat# 233-FB, R&D Systems, USA).

Techniques: Comparison, Immunostaining, Expressing, Staining, Derivative Assay, Marker, Standard Deviation

Journal: Military Medical Research

Article Title: Engineering of functional auditory neurons from human induced pluripotent stem cells

doi: 10.1016/j.mmr.2026.100008

Figure Lengend Snippet: Origin of human SGN-like neurons revealed by scRNA-seq. a Schematic illustration of the developmental origins of auditory neurons in the human inner ear in vivo . b UMAP plots of SK8-A D25 cells, color-coded as in a. c Feature plots showing key gene markers for the classification of cell subtypes and determination of neuronal origins. d Re-clustering of isolated otic neural primordium cells from b, separating into three populations: EMT, delaminating neuroblasts, and neurogenesis. e Heatmap showing the top 100 differentially expressed genes across the three distinctive clusters: EMT, NB, and NG. The X and Y axes represent differentially expressed genes and single cells, respectively. f Violin plots displaying marker gene expression in a log-transformed scale among the three different clusters of otic neural primordium. The number in the upper right corner represents log-normalized gene expression (log1p normalized UMI counts) per cell. g Feature plot visualizing expression of SGN and VGN markers in the otic neural primordium cluster, based on scRNA-seq data from previous studies . Numbers in the lower left corners indicate the percentage of cells expressing each marker. h Dot plots illustrating minimal expression of hindbrain, GG, and EPI markers, and high expression of otic neuroblast and neuronal markers. To support data sharing, visualization, and analysis, the processed scRNA-seq dataset for is available on the gEAR portal ( https://umgear.org/p?l=sk8aNeuron ). SGN. Spiral ganglion neuron; scRNA-seq. Single-cell RNA sequencing; UMAP. Uniform manifold approximation and projection; EMT. Epithelial-mesenchymal transition; NB. Neuroblasts; NG. Neurogenesis; VGN. Vestibular ganglion neurons; GG. Geniculate ganglion; EPI. Epibranchial placode; PLP1. Proteolipid protein 1; TWIST1. Twist family bHLH transcription factor 1; TOP2A. DNA topoisomerase II alpha; MEUROD1. Neuronal differentiation 1; SOX2. SRY-box transcription factor 2; WNT4. Wnt family member 4; FGF8. Fibroblast growth factor 8; DCX. Doublecortin; MEIS2. Meis homeobox 2; PROX1. Prospero homeobox 1; TLX3. T cell leukemia homeobox 3; SALL3. Spalt like transcription factor 3; PVALB. Parvalbumin; CNC. Cranial neural crest; ZBTB16. Zinc finger and BTB domain containing 16; PAX6. Paired box 6; HOXA2. Homeobox A2; FOXI1. Forkhead box I1; PHOX2A. Paired mesoderm homeobox protein 2A; FOXI2. Forkhead box I2; NEUROG1. Neurogenin 1; INSM1. Insulinoma-associated 1; HE56. Hes family BHLH transcription factor 6; MAP2. Microtubule-associated protein 2; TUBB3. Tubulin beta 3 class III.

Article Snippet: On D3, the medium was replaced with a CDM containing 10 ng/ml bone morphogenetic protein 4 (BMP4; Cat# 314-BP, R&D Systems, USA), 1 μmol/L SB-431542 (Cat# 1614, TOCRIS, UK), and 10 ng/ml basic fibroblast growth factor 2 (FGF2, Cat# 233-FB, R&D Systems, USA).

Techniques: In Vivo, Isolation, Marker, Gene Expression, Transformation Assay, Expressing, Single Cell, RNA Sequencing