Fig 1: Ocimum sanctum hydrophilic fraction‐1 (OSHP‐1) blocks the methyl tetra hydro folate (MTHF)‐mediated dedifferentiation of glial cells and promotes redifferentiation. (A): O9‐1 cells were first differentiated to glial cells for 10 days. Then they were subjected to (+) or (−) MTHF mediated dedifferentiation in the presence of 200 ng/ml OS‐AQ, OSHP‐1, OSHB‐1, and OSHB‐2. The crestospheres >50 μM size were counted (n = 6). *, p < .001; **, p < .0001, Student's t test. (B): Cell viability/cell proliferation assays were performed using MTS Assay Kit (ab197010). Two cell lines were taken: (i) O9‐1 cells and (ii) human fibroblast CRL‐2522 (ATCC). Cells were treated with vehicle or OSHP‐1 (50–300 ng/ml) for 24, 48, and 72 hours. After that the cells were washed with PBS three times and assayed for cell viability/proliferation as per the manufacturer's instructions (n = 4, each experiment done in triplicate). (C): The MTHF mediated dedifferentiated glial cells treated with OSHP‐1 in the presence or absence of MTHF or after washing away MTHF were triturated and plated on laminin coated glass slides and immunostained for GFAP and counterstained with DAPI. OSHP‐1 caused redifferentiation of spheres obtained from MTHF mediated dedifferentiated glial cells. The MTHF mediated spheres were cultured in the absence of glial differentiation media but in the presence of OSHP‐1 (200 ng/ml) for 4 days and then the cells were immunostained for GFAP, Oct4, Sox2, Klf4, and counterstained with DAPI. (D): Effect of active components of OSPH‐1, AA (AA, 250 μM), rosmarinic acid (RA, 12.5 μg/ml), and caffeoylquinic acid (CA, 125 μM) on MTHF‐mediated dedifferentiated cells to form crestospheres. (E): MTHF‐mediated dedifferentiated O9‐1 cells were treated with AA (250 μM), RA (12.5 μg/ml), CA 125 μM, and mixture of AA + RA + CA for 72 hours Western blots of the total cell lysate (RIPA lysates) and nuclear fractions. Fifteen micrograms protein per well was immunoblotted using GFAP, β‐actin (positive control), pRb (as nuclear marker control), Sox2, Oct4, and folate receptor‐α. Semiquantitative blot quantization was done by the software provided in Bio‐Rad ChemiDocMP system.
Fig 2: Characterization of ACE2 and TMPRSS2 levels and Spike protein pseudovirus entry into induced pluripotent stem cell‐derived human cardiomyocytes. A, Ai, Immunostaining of cell nuclei (blue), angiotensin converting enzyme 2 (ACE2, yellow), F‐Actin (red) and transmembrane serine protease 2 (TMPRSS2, green) in induced pluripotent stem cell‐derived human cardiomyocytes (iCMs). iCMs were derived from the DU11 induced pluripotent stem cell cell line, reprogrammed episomally from BJ fibroblasts (ATCC CRL‐2522) at the Duke University iPSC Core Facility. Aii, reverse transcription quantitative polymerase chain reaction of ACE2 and TMPRSS2 expression levels in iCMs and wild‐type 293 cells (n=3, ****P<0.0001, **P<0.01, Student t test). B, Luciferase reading of iCMs (i) and 293ACE2 (ii) cells with or without D614G Pseudo‐V. Addition of polybrene (PB) along with Pseudo‐V did not enhance viral entry into iCMs or 293ACE2 cells (n≥3, *P<0.05, based on ANOVA with post hoc Tukey's test). iCMs were transduced with 5x Pseudo‐V and 293ACE2 cells were transduced with 1x Pseudo‐V. C, Effect of inhibitors of ACE2 (Captopril, 100 μM), TMPRSS2 (Camostat, 5 μM and Nafamostat, 0.5 μM), cathepsins (E64D, 30 μM and SID26681509, 10 μM), and endosomal acidification (chloroquine, 10 μM) on D614G pseudovirus entry into iCMs and 293ACE2 cells (n≥3, *P<0.05 based on ANOVA with post hoc Dunnett's test). Drug concentrations used were based on published results 1 and preliminary studies. D, Entry of pseudovirus expressing B.1.617.2 Delta or BA.1 Omicron Spike protein into iCMs (Di) or 293ACE2 (Dii) as detected by chemiluminescence (n=6, *P<0.05, **P<0.01 based on Student t test). E, Ei, schematic of the experimental timeline for viral infection of 3‐dimensional (3D) cardiobundles before bundle assembly. Drug (NT: not treated vehicle control) was first added to the cells for 30 min and then Pseudo‐V was added to cells for another 60 min. Then the cells were poured onto the molds for fabrication of cardiobundles. Eii and Eiii, Luciferase reading of 3D cardiobundles treated with chloroquine, 10 μM (Eii) or E64D, 30 μM (Eiii) infected with Delta or Omicron compared with NT (n≥3, ****P<0.0001 based on 2‐way ANOVA test with post hoc Tukey's test). Schematic of the experimental timeline was created with BioRender.com. F, Fi, schematic of the timeline for viral infection and drug treatment of cardiobundles. 3D cardiobundles were first fabricated and matured for 6 days. Drug (NT: vehicle) was added to the medium at day 6 for the following 4 days and virus was added to the medium at day 7 for the following 3 days. Fii, Luciferase reading of cardiobundles treated with chloroquine, 10 μM (Eii) or E64D, 30 μM (Eiii) infected with Delta or Omicron compared with NT (n=4, ****P<0.0001, ***P<0.001 based on 2‐way ANOVA test with post hoc Tukey's test). Schematic of the experimental timeline was created with BioRender.com.
Fig 3: BJ cell line human fibroblasts (a). cultured in the flask (intra-experiment), (b). in a 96-well plate for cytotoxicity evaluation with the MTT Assay (intra-experiment). (c,d) Pre-experiment BJ cell line fibroblasts as observed under a microscope. Cells were obtained from ATCC (CRL-2522). Image (c,d) courtesy: ATCC, https://www.atcc.org/products/crl-2522#product-references (accessed on 27 October 2024).
Fig 4: Apoptotic responses measured spectrophotometrically by OD at 570 nm (formazan absorption) induced by the compounds studied in human foreskin fibroblasts HFF-BJ (ATCC CRL-2522 cells). Cells were treated with the indicated concentrations of compounds 1 to 14 (A) and compounds 15 to 21 (B) for 72 h. Cell viability as visualized by fold change of formazan-positive cells normalized to the untreated control and DMSO. Viability reduced below 40% represents the cytotoxic zone, below the dotted line in each plot. The number of biological replicates is indicated.
Fig 5: Effects of pharmacologically induced polyploidization on human induced pluripotent stem cell–derived cardiomyocytes.hiPSC‐CMs were derived from the DU11 hiPSC line, reprogrammed episomally from BJ fibroblasts (ATCC CRL‐2522) at Duke University, via a widely used 2D, WNT‐based protocol that generates beating CMs by Day 6 to 7 of differentiation, which are then purified via lactate supplementation in the absence of glucose, resulting in ≈90% cardiac troponin T + CMs that are primarily ventricular, with no detectible MLC2a+/MLC2v‐ CMs (as we showed in Shadrin et al. Nat Comm, 2017). A, Ctrl (DMSO‐treated) and AURORA Kinase B inhibitor (AURKBi) (AZD1152‐treated; 100 nmol/L for 4 days starting on days 16 to 20 post‐initiation of CM differentiation) hiPSC‐CMs were analyzed via flow cytometry for Hoechst 33342 fluorescence intensity to quantify the abundance of polyploid (>2N DNA content) cells (n=66, Welch's t test). AURKBi had no effect on CM purity or MLC2a/v expression (data not shown). B, Representative immunofluorescence image and associated quantification of hiPSC‐CM area following AURKBi treatment (n=8–9, Student t test) with accompanying analysis of flow cytometry of hiPSC‐CMs showing differences in cell size either by ploidy within each treatment group (left of vertical dotted line, n=60, 2‐way ANOVA with Holm‐Sidak post‐hoc tests) or by treatment group population (right of vertical dotted line; n=60, Welch's t test) and (B′) relative cleaved caspase 3 (Cc3) staining for apoptotic cells (n=12, Student t test). (C–C″) Bulk RNA‐sequencing of control and AURKBi hiPSC‐CMs from 5 differentiation batches showing resulting volcano plot (C) followed by (C′) GO analysis with top and selected GO terms for biological processes (based on FDR <0.05 and Log2FC >0.5 or <−0.5) and (C″) heat maps for selected important cellular processes and the changes in associated gene expression. D, Flow cytometry of hiPSC‐CMs showing quantifications of transcription rate (5‐ethynyl uridine incorporation staining intensity; n=11–12), translation rate (L‐homopropargylglycine incorporation staining intensity; n=8–9), nucleolin staining intensity (n=9), and mitochondrial abundance (Mitotracker dye intensity; n=12) for the total cell population (right of vertical dotted lines, Student t test) and separated by ploidy within each treatment group (left of vertical dotted lines; same n's as total population data with 2‐way ANOVAs with post‐hoc Holm‐Sidak's test). E, Schematic showing treatment and EdU dosing in hiPSC‐CMs. E′–E″, Quantifications from flow cytometry (E′) and immunofluorescence (E″) analysis of EdU incorporation rates and AURKB expression (staining the whole nucleus [left] or cleavage furrow [right]) within control and AURKBi hiPSC‐CMs additionally treated with either DMSO (+Veh) or 2 μmol/L CHIR99021 (+CHIR) along with 10 μmol/L EdU for 48 h before sample collection (E′ n=9 with 2‐way ANOVA with post‐hoc Fisher's LSD test, E″ n=6 with 2‐way ANOVA with post‐hoc Fisher's LSD test) with accompanying heat map derived from RNAseq analysis in (C) showing relative expression of cell cycle–related genes. (F) Representative traces of Cal520‐AM dye fluorescence in hiPSC‐CM monolayers paced at 2.5 Hz and corresponding quantifications of Ca2+ transient amplitude (n=16–22 Student t test) and kinetics (n=16–18, Student t test). F′, Representative traces of Cal520‐AM dye fluorescence in hiPSC‐CM monolayers during application of 40 mmol/L bolus caffeine and corresponding quantifications of Ca2+ transient amplitude, rate constant of Ca2+ efflux via Na+/Ca2+ exchanger (kNCX, n=8–10, Student t test), and rate constant of SR Ca2+ uptake via SERCA (kSERCA, n=8–10, Student t test). All n's are defined as individual CM monolayers, and experiments were conducted in at least 3 differentiation batches, with assessment of polyploidy in A' being from 22 differentiation batches. Outliers were excluded from data analysis using ROUT method with Q=1. Data analysis was confirmed by at least 1 author blinded to experimental groups. Quantifications of calcium imaging were performed using custom Matlab scripts. Cardiac troponin T abundance (≈90% cardiac troponin T+) was not affected by AURKBi treatment (data not shown). Beeswarm plots are shown with individual data points and mean±SEM, and violin plots are depicted for n >22 showing distribution of all data points. RNAseq data were mapped with the GRCh38 reference genome assembly (Cunningham et al., Nucleic Acids Res. 2022) using HISAT2 (Kim et al., Nature Biotechnology 2019). Differential gene expression analysis was performed with the edgeR (Robinson et al., Bioinformatics 2009) package in R. The volcano plot was generated with the EnhancedVolcano package in R (Blighe, Rana, and Lewis, 2018) and GO analysis was performed with the clusterProfiler package in R (T. Wu et al., The Innovation 2021). GO indicates gene ontology; hiPSC‐CMs, human induced pluripotent stem‐cell‐derived cardiomyocytes; and LSD, Fisher's Least Significant Difference test.
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