Journal: Aging and Disease

Article Title: Brain Resident Ly6C hi Monocytes Are Necessary for Maintaining Adult Hippocampal Neurogenesis

doi: 10.14336/AD.2024.0835

Figure Lengend Snippet: Microglia activation is not involved in stress-induced inhibition of AHN . ( A ) Hippocampal coronal sections of the brains of mice in the WT and APP/PS1 groups were stained with Iba1 antibody. Scale bar = 100 μm, Scale bar of morphological analysis = 20 μm. (B, C, D, E) Quantification of the density of Iba1 + microglia (B), average body area (C), number of endpoints (D), and average process length of microglia (E). n = 3 mice in each group. ( F ) Hippocampal coronal sections of the brains of mice in the PBS i.c.v. and LPS i.c.v. groups were stained with Iba1 antibody. Scale bar = 100 μm, Scale bar of morphological analysis = 20 μm. (G, H, I, J) Quantification of the density of Iba1 + microglia (G), average body area (H), number of endpoints (I), and average process length of microglia (J). n = 5 mice in each group. ( K ) Hippocampal coronal sections of the brains of mice in the NS, LH and NLH groups were stained with Iba1 antibody. Scale bar = 100 μm, Scale bar of morphological analysis = 20 μm. (L, M, N, O) Quantification of the density of Iba1 + microglia (L), average body area (M), number of endpoints (N), and average process length of microglia (O). n = 5-6 mice in each group. ( P ) Hippocampal coronal sections of the brains of mice in the NS and CRS groups were stained with Iba1 antibody. Scale bar = 100 μm, Scale bar of morphological analysis = 20 μm. (Q, R, S, T) Quantification of the density of Iba1 + microglia (Q), average body area (R), number of endpoints (S), and average process length of microglia (T). n = 5 mice in each group. For (B-E, G-J, L-O, Q-T), data are represented as mean ± SEM. Statistical analysis was performed using Kruskal-Wallis test (L-O), Mann Whitney test (B-E, G-J) and two-tailed unpaired t-tests (Q-T); *p < 0.05, **p < 0.01.

Article Snippet: Rabbit anti-mouse Iba1 , proteintech , 10904-1-AP.

Techniques: Activation Assay, Inhibition, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Aging and Disease

Article Title: Brain Resident Ly6C hi Monocytes Are Necessary for Maintaining Adult Hippocampal Neurogenesis

doi: 10.14336/AD.2024.0835

Figure Lengend Snippet: Depletion of circulating monocytes reproduced the inhibitory effects of AHN by stress . ( A ) Hippocampal coronal sections of the brains of mice in the PBS i.v. and CLO i.v. groups were stained with DCX antibody. Scale bar = 100μm, Scale bar of morphological analysis = 100 μm. ( B ) Quantification of the density of DCX + newborn immature neuron, average dendritic branch number and average dendritic branch length. n = 6 mice in each group. ( C ) Hippocampal coronal sections of the brains of mice in the PBS i.v. and CLO i.v. groups were stained with Iba1 antibody. Scale bar = 100μm, Scale bar of morphological analysis = 20 μm. ( D ) Quantification of the density of Iba1 + microglia, average body area, number of endpoints and average process length of microglia. n = 6 mice in each group. For (B, D), data are represented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-tests; ****p < 0.0001.

Article Snippet: Rabbit anti-mouse Iba1 , proteintech , 10904-1-AP.

Techniques: Staining, Two Tailed Test

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) HLA class I and II expression in HIP-NILB-iPSCs with or without IFN-γ treatment. Isotype is a negative control with matched primary antibody. (B) PBMCs cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (C) ELISA assay for IFN-γ secretion in PBMCs cocultured with HIP-NILB-iPSCs. (D) CFSE analysis of T cells (CD3 + ) proliferation in PBMCs when cocultured with HIP-NILB-iPSCs. PBMCs is negative control (Neg.), PBMCs activated by PHA is positive control (Pos.). (E) Primary NK cells cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (F) ELISA assay for IFN-γ secretion in Primary NK cells cocultured with HIP-NILB-iPSCs. (G) NK degranulation assay by quantifying CD107a surface expression in CD56 + NK cells cocultured with HIP-NILB-iPSCs. NK cells alone as Neg., NK cells treated with PHA as Pos.. (H) Activation of HMC3 cells (CD68 + ) in IBA1 + when incubated with HIP-NILB-iPSCs. HMC3 alone as Neg., LPS-stimulated HMC3 as Pos.. (I) HMC3 cells cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (J) The phagocytic activity of THP-1 derived macrophages against HIP-NILB-iPSCs is characterized by the percentage of double positive of Deep Red and FITC. (K) Gene set enrichment analysis (GSEA) shows the enriched signaling pathway in HIP-NILB-iPSCs against NILB-iPSCs. NES represents the normalized enrichment score. All experiments were independently duplicated three times. Data are mean ± SEM, p values were determined using a two-tailed, unpaired Student’s t-test (b, c, e, f, g, h, i, j) or Welch’s t-test (D), * 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: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Degranulation Assay, Activation Assay, Incubation, Activity Assay, Derivative Assay, Two Tailed Test

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: Representative images showing lower magnification images of TDP-43 in TUJ1 + neurons in WT, ALS and HIP-NILB-iPSCs treated pigs (correspond to ). Scale bar, 200 μm. (B-G) Representative images showing lower magnification images of (B) CHRNA7 (correspond to ), (C) SYN (correspond to ), (D) Vimentin (correspond to ), (E) MBP (correspond to ), (F) NLRP3 (correspond to ), (G) IBA1 (correspond to ), respectively, in the lumbar spinal cord of WT, ALS and HIP-NILB-iPSCs treated pigs. Scale bar, 200 μm.

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:

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) Representative immunofluorescence images of a-BTX/NF-H/GFP/DAPI showing reformed neuromuscular junctions between HIP-NILB-iPSCs-derived MNs and host gastrocnemius muscle fiber in Pig-2# and Pig-3# (three and four months after grafting), but not in Pig-1# (two months after grafting), WT or ALS controls. Scale bar, 10 μm. (B-H) Representative immunofluorescence images for (B) CHRNA7, (C) SYN, (D) Vimentin, (E) MBP, (F) NLRP3, (G) IBA1 and (H) GFAP in the lumbar spinal cord of WT, ALS and HIP-NILB-iPSCs treated pigs, and their quantifications (n=3). Scale bars, 100 μm or 50 μm. (I) Western blot analysis of CHRNA7, Vimentin, MBP, NLRP3 and GFAP in lumbar spine cord of WT, ALS and HIP-NILB-iPSCs treated pigs. The statistical analysis was performed by one-way ANOVA following Tukey’s multiple comparisons. Data was shown as 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: Immunofluorescence, Derivative Assay, Western Blot

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

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) Representative images showing lower magnification images of TDP-43 corresponding to . Scale bar, 100 μm. (B-G) Representative images showing lower magnification images of (B-E) CHRNA7, SYN, Vimentin and MBP (corresponding to ), and (F-G) IBA1 and NLRP3 (corresponding to ), respectively. Scale bars, 200 μm or 100 μm.

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: