mouse primary embryonic fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Enhanced liver regeneration via targeted mRNA delivery for partial in vivo reprogramming

doi: 10.1101/2025.11.03.686120

Figure Lengend Snippet: (a) Illustration of C12-SPM-GAL LNP-mRNA formulation. Chemical structure of the ionizable lipid C12-SPM and the lipid components with their respective molar ratios in the optimized C12-SPM-GAL LNP-mRNA formulation. (b) Biochemical assays for serum ALT and AST levels (n = 3) 24 h after intravenous (IV) administration of LNPs loaded with firefly luciferase mRNA (0.3 mg/kg) in C57BL/6 mice. PBS; PBS-injected mice, MC3; DLin-MC3-DMA LNP-injected mice, GAL; C12-SPM-GAL LNP-injected mice. (c) Time-course analysis of in vivo bioluminescence following IV administration of LNPs loaded with firefly luciferase mRNA in C57BL/6 mice (0.3 mg/kg) (n = 3 ∼ 4). (d) Detection of OSKM proteins 24 h after transfection of C12-SPM-GAL LNPs loaded with OSKM mRNA into MEFs (1 μg total OSKM mRNA for 100,000 cells, 0.25 μg for each mRNA). NT; no treatment, OSKM; OSKM mRNA-LNP treatment. (e) Experimental outline for assessing in vivo partial reprogramming by OSKM mRNA-LNP to induce hepatocyte dedifferentiation in normal wild-type mice. The PBS-injected group (PBS group) was used as a negative control, and both Luc mRNA-LNP (Luc group) and OSKM mRNA-LNP (OSKM group) were IV administered at a dose of 0.3 mg/kg. (f) Quantitative PCR analysis of liver progenitor-like cell (LPLC)-related genes in the liver 30 h post-injection (n = 4 ∼ 5). (g) Immunofluorescence staining of Sox9 and Ki-67 co-stained with Hnf4α in the liver 30 h post-injection. DAPI stains the nuclei (scale bars = 100 μm). (h) Western blot analysis for LPLC marker Sox9, cell cycle marker phosphorylated histone H3 (pHH3), and proliferating cell nuclear antigen (PCNA) in the liver 30 and 54 h post-injection. (i) Schematic illustration of C12-SPM-GAL-mediated OSKM mRNA treatment in an acetaminophen (APAP)-induced acute liver injury mouse model. C57BL/6 mice received intraperitoneal injection of APAP at a dose of 400 mg/kg after fasting for 12 h. Mice were treated with PBS (Mock group), Luc mRNA-LNP (Luc group), or OSKM mRNA-LNP (OSKM group) 18 h post-APAP administration. (j) Kaplan-Meier survival plot of mice (n = 14) in each treatment group over four days following APAP administration. Cont; normal mice, Mock; APAP injury + PBS-injected mice, Luc; APAP injury + Luc mRNA-LNP-injected mice, OSKM; APAP injury + OSKM mRNA-LNP-injected mice. (k) Blood chemistry analysis of serum AST and ALT levels 96 hours post-APAP administration (78 hours post-treatment). (l) Representative images of the whole livers and H&E staining 96 hours post-APAP administration (78 hours post-treatment) (left panel) (scale bars = 500 μm). Dotted lines indicate necrotic areas. Quantification of the necrotic area in three H&E-stained sections per mouse liver sample (right panel) (n = 4 mice per group). (m) Immunostaining for cleaved caspase-3 (cCasp3) (red) in the pericentral region of the liver, with nuclei counterstained with DAPI (blue) (scale bar = 100 μm). CV indicates the central vein. (n) Western blot analysis for cleaved caspase-3 (cCasp3) 96 h post-APAP administration (78 h post-LNP injection at indicated mRNA doses). Vinculin was used as an internal control. Statistical analysis was performed using one-way ANOVA: p < 0.01(**), p < 0.001(***), ns, not significant.

Article Snippet: Primary mouse embryonic fibroblasts (MEFs) were isolated from ICR mouse embryos at E13.5 days (Orient Bio) as previously described .

Techniques: Formulation, Luciferase, Injection, In Vivo, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Western Blot, Marker, Immunostaining, Control