Journal: bioRxiv
Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models
doi: 10.1101/2025.09.03.673895
Figure Lengend Snippet: (A) Nissl staining of lumbar spinal cord in WT, ALS and HIP-NILB-iPSCs treated rabbits, and representative magnified images for ventral horn area (n=3). Scale bars, 1 mm or 100 μm. (B) Quantifications of gray to white matter ratio and average neuron number in the unilateral ventral horn (n=3). (C) Western blot analysis of NeuN expression in spinal cords of WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (D) Quantitation of STEM121 + ChAT + human MNs and STEM121 - ChAT + rabbit MNs cell numbers in the lumbar spinal cord of three HIP-NILB-iPSCs treated rabbits. WT and ALS rabbits were used as controls. (E) Representative immunofluorescence images displaying nuclear localization of TDP-43 in TUJ1 + neurons in HIP-NILB-iPSCs treated. WT and ALS rabbits were used as controls. Scale bar, 50 μm. (F) Western blot analysis of the soluble and insoluble TDP-43 in supernatants and pellets of rabbit lumbar spinal cords. (G) ELISA assay for serum glutamate concentration in WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (H) Representative H&E and Masson staining images showing the atrophy and fibrosis of gastrocnemius muscle in WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). Scale bar, 100 μm. (I) Creatine kinase activity in the serum of WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (J) Representative EMG signals showing spontaneous activities in gastrocnemius muscle of WT, ALS and HIP-NILB-iPSCs treated rabbits. (K) Gait analysis and quantification of stride and sway length in hind limbs of WT (n=4), ALS (n=5) and HIP-NILB-iPSCs treated rabbits (n=5) at two months after grafting. (L) Analysis of tension index of hind limbs over time in WT (n=4), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). The statistics analysis was performed with two-way ANOVA followed by Tukey’s multiple comparisons, and the significant differences were labeled between ALS and HIP-NILB-iPSCs group in different time points. (M) Representative immunofluorescence staining of CHRNA7, SYN, Vimentin and MBP in lumbar spinal cord of WT (n=3), ALS (n=4) and HIP-NILB-iPSCs treated rabbits (n=3), and their quantifications. Scale bar, 100 μm. (N) Representative immunofluorescence staining of NLRP3, IBA1 and GFAP in lumbar spinal cord of WT (n=3), ALS (n=4) and HIP-NILB-iPSCs treated rabbits (n=3), and their quantifications. Scale bars, 100 μm or 50 μm. (O-P) (O) Western blot analysis and (P) Real-time PCR analysis of CHRNA7, SYN, Vimentin, MBP, NLRP3, IBA1, GFAP in lumbar spinal cords of WT (n=3), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). (Q-R) Real-time PCR analysis of (Q) neurotrophic factors ( NTF3 , NTF4 , NGF , BDNF ) and (R) inflammatory factors ( IFNG , IL17A , CD86 , CD204 and TGFB2 ) in lumbar spinal cords of WT (n=3), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). The statistical data were analyzed by one-way ANOVA followed by Dunnett’s T3 multiple comparisons (N-NLRP3) or Tukey’s multiple comparisons (b, d, g, i, k, m, n-IBA1, n-GFAP, p, q, r) unless mentioned otherwise. Data was mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S, not significant.
Article Snippet: The primary antibodies were used as follows: chicken anti-GFP polyclonal antibody (ThermoFisher, A10262, 1:300), mouse anti-GFP monoclonal antibody (Proteintech, 66002-1-Ig, 1:300), rabbit anti-GFP polyclonal antibody (Proteintech, 50430-2-AP, 1:300), mouse anti-beta III Tubulin (TUJ1) monoclonal antibody (Abcam, ab78078, 1:500), mouse anti-MAP2 monoclonal antibody (Sigma-Aldrich, M1406, 1:300), Rabbit anti-Choline Acetyltransferase (ChAT) monoclonal antibody (Sigma-Aldrich, ab181023, 1:300), mouse anti-GFAP monoclonal antibody (Santa Cruz, sc-33673, 1:1000), mouse anti-Nuclei monoclonal antibody (Millipore, MAB4383, 1:200), mouse STEM121 antibody (Takara, Y40410, 1:400), mouse anti-Neurofilament 200 monoclonal antibody (Sigma-Aldrich, N5389, 1:400), α-Bungarotoxin, Alexa Fluor® 647 Conjugate (ThermoFisher, B35450, 1:1000), rabbit anti-CHRNA7 polyclonal antibody (Proteintech, 21379-1-AP, 1:200), rabbit anti-Synaptophysin (Syn) monoclonal antibody (Abcam, ab52636, 1:200), rabbit anti-Vimentin polyclonal antibody (Proteintech, 10366-1-AP, 1:200), rabbit anti-Myelin basic protein (MBP) polyclonal antibody (Proteintech, 10458-1-AP, 1:200), rabbit anti-NLRP3 polyclonal antibody (Proteintech, 19771-1-AP, 1:200), rabbit anti-IBA1 polyclonal antibody (Proteintech, 10904-1-AP, 1:500), rabbit anti-TDP43 polyclonal antibody (Proteintech, 10782-2-AP, 1:200).
Techniques: Staining, Western Blot, Expressing, Quantitation Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activity Assay, Labeling, Real-time Polymerase Chain Reaction
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