Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, After gene editing, LMNB1-edited clones #5 and #8 were established as cell lines, expanded, and their normal karyotype confirmed. b-c, Western blot and immunocytochemical analysis of gene-edited and clone cell line demonstrating expression of typical markers for pluripotent (OCT4) and differentiated cells (Brachyury for mesoderm; SOX17 for endoderm; PAX6 for ectoderm) after directed differentiation in adherent cultures. Scale bar in c : 50 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Clone Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, EB formation in the presence of DMSO, Y-27632 and CEPT. Human ESCs (WA09) cells were dissociated with Accutase and plated into 6-well ULA plates in E6 medium. Representative phase-contrast images were taken at 24 h post-plating. Scale bar, 50 μm. b, To generate single EBs, hESCs were dissociated with Accutase and plating into AggreWell plates (5,000 cells/well). Images were taken 24 h post-plating. In the presence of DMSO the vast majority cells underwent cell death and EB formation was not observed. Treatment with Y-27632 supported EB formation but significant numbers of dead cells were detected surrounding the EB. Note that CEPT enables EB formation without apparent cell death. Scale bar, 100 μm. c, Quantification of the diameter of single EBs (24 h post-plating). Data are mean ± s.d. (n = 20 EBs for Y-27632 and n = 22 for CEPT), **** P < 0.0001, two-tailed Student’s t -test. d, Single EB formation in 96-well ULA plates. Dissociated hESCs were plated into 96-well ULA plates at 2,000 cells/well. Live and dead cells were stained with calcein green AM and propidium iodide (PI) 24 h after cell seeding. Scale bars, 100 μm. e, Quantification of cell survival in single EBs at day 1 and day 7 by using the CTG 3D assay. Note the significant difference between Y-27632 and CEPT treatment at both timepoints. Data represent mean ± s.d. (n = 24 EBs for each group), **** P < 0.0001, one-way ANOVA. f, CEPT supports differentiation of single EBs into the three germ layers. Individual EBs were cultured in E6 medium to allow for spontaneous differentiation and analyzed on day 7 for the expression of PAX6, SOX17, and Brachyury using an optimized quantitative RT-PCR protocol that enabled detection of low transcript levels in single EBs (see Materials and Methods section for details). Data represent mean ± s.d. (n = 3 experiments and in each experiment 24 EBs were analyzed for each group), * P = 0.0327, two-tailed Student’s t -test. g, h, Cerebral organoids were generated by using Y-27632 and CEPT for the first 24 h. At day 30, organoids were fixed, sectioned, processed for histology (hematoxylin and eosin stain) and immunohistochemistry for FOXG1. Representative images show that CEPT treatment resulted in larger organoids and more abundant FOXG1-expressing cells. Scale bars, 400 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Two Tailed Test, Staining, Cell Culture, Expressing, Quantitative RT-PCR, Generated, H&E Stain, Immunohistochemistry

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: Basal expression levels of Caspase-3 in hESCs (WA09) in comparison to their lineage-restricted precursors after directed differentiation into ectoderm (PAX6), mesoderm (Brachyury), and endoderm (SOX17). Note the strong Caspase-3 expression at the pluripotent state and downregulation upon differentiation.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Expressing, Comparison