Journal: Alzheimer's & Dementia

Article Title: Human iPSC‐derived GABAergic interneuron transplantation restores circuit balance and cognitive function in an Alzheimer's disease model

doi: 10.1002/alz.71378

Figure Lengend Snippet: Generation of hiPSC‐derived MGE‐pINs. (A) Schematic illustration of generation of iPSC‐derived MGE‐pINs. (B) Immunocytochemistry of iPSC‐derived MGE progenitor cells after 3 weeks of differentiation (from three independent iPSC lines). Scale bar = 50 µm. (C) Immunocytochemistry of 5‐week‐old pINs derived from iPSCs after 2 weeks of treatment with MEK inhibitor starting at 3 weeks of differentiation. Scale bar = 50 µm. (D) Immunocytochemistry of 7‐week‐old pINs derived from iPSCs after 2 weeks of treatment with MEK inhibitor combined with CDK and NOTCH inhibitors starting at 5 weeks of differentiation. Scale bar = 50 µm. (E) Quantitative analysis of 3‐week‐old MGE progenitor cells derived from iPSCs. (F) Quantitative analysis of 5‐week‐old pINs derived from iPSCs. (G) Quantitative analysis of 7‐week‐old pINs derived from iPSCs. Data were presented as mean ± SEM ( n = 3 hiPSC lines). ERBB4, Erb‐B2 receptor tyrosine kinase 4; FOXG1, Forkhead box G1; GABA, gamma‐aminobutyric acid; hiPSC, human induced pluripotent stem cell; KI67, Ki‐67 antigen; LHX6, LIM homeobox 6; LHX8, LIM homeobox 8; MAF, v‐maf musculoaponeurotic fibrosarcoma oncogene homolog; MAFB, v‐maf musculoaponeurotic fibrosarcoma oncogene homolog B; MGE, medial ganglionic eminence; NKX2‐1, NK2 homeobox 1; OCT4, octamer‐binding transcription factor 4; SOX2, SRY‐box transcription factor 2; SOX6, SRY‐box transcription factor 6.

Article Snippet: From week 5 to week 7, in addition to the continued treatment with the MEK inhibitor, NOTCH inhibitors (DAPT 10 μM; HY‐13027, MCE) and cyclin‐dependent kinase (CDK) inhibitors (PD0332991 2 μM; MCE, HY‐50767) were introduced to induce cell cycle exit in the cells during this stage.

Techniques: Derivative Assay, Immunocytochemistry, Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Potential effects of endogenous RNA/DNA hybrids on CRISPR-Cas9-mediated homology-directed repair

doi: 10.1016/j.omtn.2026.102880

Figure Lengend Snippet: Modulation of HDR efficiency by R-loop resolution, cell-cycle perturbation, and locus context (A) ddPCR quantification of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or 10,000 vg/cell of scAAVDJ-sg3′ Alb + 10,000 vg/cell ssAAVDJ-donor 3′ Alb. (B) Schematic representation of cell cycle profiles in HEPA1-6 cells and murine hepatocytes. (C) Flow cytometry analysis of HEPA1-6/SpCas9 cells treated for 24 h with rapamycin (1 μM, 5 μM) or palbociclib (PD; 5 μM, 10 μM). DMSO-treated cells served as controls. (D) DRIP-qPCR analysis of HEPA1-6/SpCas9 cells after 24 h treatment with 5 μM rapamycin; DMSO-treated cells served as controls. (E) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or scAAVDJ-sg3′ Alb + ssAAVDJ-donor 3′ Alb. (F) IGV visualization of the Alb and Actb loci showing gRNA (sgActb) and donor (donor Actb) design targeting the 5′ region of Actb . (G) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transduced for 72 h with 10,000 vg/cell of scAAVDJ-sgActb + 10,000 vg/cell ssAAVDJ-donor Actb. Scrambled gRNA with donor served as negative control. (H) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (I) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (J) DRIP-qPCR analysis of CD4 + T cells from a healthy donor, pre- and post-activation with 10 μg/mL PHA, 50 IU/mL IL-2, 5 ng/mL IL-7, and 5 ng/mL IL-15. DRIP, immunoprecipitated samples treated with S9.6 antibody which are enriched in R loops; RNAseH + , samples treated with RNAseH1+S9.9 antibody which are depleted of R-loops. (K) Table summarizing the effects of R-loops levels on indels, HDR, and AAV integration. N/A, not affected; ↑ increase; ↓ = decrease. Statistical analysis: (A and E) multiple t test; (C) two-way ANOVA with Dunnett’s post hoc test; (G–I) Student’s t test. p < 0.05, ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001. Error bars represent mean ± SD.

Article Snippet: HEPA1-6/SpCas9 cells were treated with rapamycin (Selleck, S1039) or palbociclib (Selleck, S1116) for 24 h, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry (NovoCyte Penteon, Agilent).

Techniques: Transduction, Flow Cytometry, Negative Control, Activation Assay, Immunoprecipitation