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(A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , <t>IgG1-Fc</t> , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.
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(A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , <t>IgG1-Fc</t> , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.
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(A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , <t>IgG1-Fc</t> , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.
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(A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , IgG1-Fc , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.

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) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , IgG1-Fc , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.

Article Snippet: The antibodies used were listed as follows: PE anti-human HLA-A,B,C (BioLegend, 311405, 1:20), PE anti-human HLA-DR,DP,DQ (BioLegend, 361716, 1:20), PE Mouse IgG2a, κ Isotype Ctrl (FC) antibody (BioLegend, 400213, 1:20), PE anti-human CD3 (BD Biosciences, 347347,1:50), FITC anti-human CD107a (BD Biosciences,555800,1:100), FITC Mouse IgG1, κ Isotype Control RUO (BD Biosciences, 555748,1:100), APC anti-human CD56 (BioLegend, 362504, 1:20), PE-Cy5 human CD56 (BD Biosciences, 555517, 1:10), APC Mouse IgG1, κ Isotype Ctrl (FC) antibody (BioLegend, 400122, 1:20), PE anti-mouse CD68 (BioLegend, 137014, 1:100), PE Rat IgG2a, κ Isotype Ctrl (BioLegend, 400508, 1:100), CoraLite® Plus 647-conjugated IBA1 monoclonal antibody (Proteintech, CL647-66827,1:200), CoraLite® Plus 647 Mouse IgG2a Isotype Control (Proteintech, CL647-65244,1:200).

Techniques: Multiplex Assay, Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Transformation Assay, Labeling, Control, Expressing, Two Tailed Test