ectocervical primary cell medium  (Thermo Fisher)

Journal: Nature Communications

Article Title: Modelling Chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming

doi: 10.1038/s41467-022-28569-1

Figure Lengend Snippet: a Schematic representation of human ectocervical organoid and 2D stem cell culture generation from biopsies. b – d Confocal images of human ectocervical tissue and organoids immunolabeled for KRT5, KRT8 (b); Ki67, E-cadherin (CDH1) ( c ); Ki67, p63 ( d ); Nuclei in blue. e PCR to detect HPV16, HPV18 E6E7, and GAPDH in samples from three independent experiments. Donor 1–3, untreated ectocervical stem cells; mock, Polybrene treated cells; Donor 1-3 HPV E6E7, Donor 1-3 transduced with HPV16 E6E7 lentivirus; End1, End1 cells; HeLa, HeLa cells; Donor 3 Luci, cells transduced with Luciferase lentivirus; J23T3, feeder cells; water, water control. f , g Confocal images of ectocervical organoids expressing HPV16 E6E7 immunolabeled for KRT5, KRT8 ( f ); Ki67, CDH1 ( g ); Nuclei are shown in blue. Images in ( b – d ) and ( f , g ) represent organoid cultures from three biological experiments. h Lysates of hCEcto and hCEcto E6E7 organoids were subjected to immunoblot analysis for CDH1 and β-actin as a loading control. Data represent three biological replicates. Densitometry values for CDH1 normalized to the β-actin, values represent the relative FC in hCEcto E6E7 compared to hCEcto. i , j Proliferation and stemness were measured by quantification of the diameter of hCEcto and hCEcto E6E7 organoids ( i ) and organoid formation ( j ). Data are presented as violin plot min–max, lines are 25 and 75th percentile, bold line is median ( i ); data are presented as mean values ± SD of four biological replicates ( j ). k Relative mRNA expression of HPV16 E6E7 analyzed by qRT-PCR from RNA extracts of three Donors (1–3) HPV E6E7, W12-Int or W12-Epi, and KC-Epi cells. Whiskers are min–max, box is 25–75th percentile, lines are median values normalized to the geometric mean of Donor 1–3 HPV E6E7. l – n smRNA-ISH images of organoids derived from hCEcto ( l ), hCEcto E6E7 ( m ), and KC-Epi ( n ) probed with HPV16 L1 and E7; Nuclei are shown in blue. Images are representatives of three replicates. Statistical significance was calculated using ( i , j ) two-sided t test, ( k ) One-way ANOVA, P -values are indicated. Source data are provided as a Source Data file.

Article Snippet: Primary cells were expanded in collagen-coated (Sigma, #C3867) tissue culture flasks until they reached 70–80% confluence in human ectocervical primary cell medium (Advanced DMEM/F-12 (Invitrogen, 12634) supplemented with 12 mM HEPES, 1% GlutaMax, 1% B27, 1% N2, 10 ng/mL human EGF (Invitrogen, 15630-056, 35050-038, 17504-044, 17502048, PHG0311), 0.5 μg/mL hydrocortisone (Sigma, H0888-1G), 100 ng/mL human noggin (Peprotech, 120-10 C), 100 ng/mL human FGF-10 (Peprotech, 100-26-25), 1.25 mM N-acetyl-L-cysteine (Sigma, A9165-5G), 2 μM TGF-β receptor kinase Inhibitor IV (SB431542, Calbiochem), 10 μM ROCK inhibitor (Y-27632) (Sigma, Y0503), 10 mM nicotinamide (Sigma, N0636), 10 μM forskolin (Sigma, F6886) before seeding ~20,000 cells/50 μL in Matrigel (BD, #356231) for culturing organoids or for maintenance of 2D stem cells on lethally irradiated J2-3T3 fibroblast feeder cells.

Techniques: Stem Cell Culture, Immunolabeling, Transduction, Luciferase, Control, Expressing, Western Blot, Quantitative RT-PCR, Derivative Assay

Journal: Nature Communications

Article Title: Modelling Chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming

doi: 10.1038/s41467-022-28569-1

Figure Lengend Snippet: a Heatmap of DEG in hCEcto and hCEcto E6E7 2D stem cells with or without Ctr infection; Columns represent biological replicates. b Venn diagram shows genes regulated in hCEcto 2D stem cells after Ctr infection or HPV E6E7 expression. Red box highlights genes upregulated by HPV E6E7 and downregulated by Ctr. c Heatmap showing GSEA enrichment (–log10( P -value)) of genes that share Cis-regulatory motifs for transcription factors. d – g hCEcto and hCEcto E6E7 2D stem cells were infected with Ctr for 48h. mRNA expression of RB1 (d), E2F1 ( e ), and TP53 ( f ) analyzed by qRT-PCR. Data represent mean ± SD from three biological replicates. Statistical significance was calculated using two-sided t test, P -values are indicated. ( g ) Immunoblot analysis for indicated proteins; β-actin. Densitometry values for E2F1 normalized to β-actin; representing FC compared to hCEcto. Data represent three biological replicates. h Dot plot showing up-or down-regulated KEGG pathways among DEG from hCEcto and hCEcto E6E7 2D stem cells with or without Ctr infection. Red arrows highlight DNA repair pathways. Dot diameter refers to the gene ratio within a group. Fill color depicts the adjusted P -value. i Heatmap of genes upregulated in ectocervical stem and differentiated cells from ref. correlated with expression profiles of hCEcto and hCEcto E6E7 organoid with or without Ctr infection. j Heatmap showing expression of genes associated with GO terms for cornification, epidermal cell differentiation, keratinization, skin and epidermis development in hCEcto and hCEcto E6E7 organoids with or without Ctr infection. k Heatmap of DEG from hCEcto E6E7 organoids with or without Ctr infection and their corresponding expression in normal and CIN1-3 tissues. The color bar depicts expression values after subtracting the mean of uninfected hCEcto samples ( i , j ) and normal cervical tissue samples ( k ) and dividing by the SD of each probe. l , m Confocal images of hCEcto 2D stem cells ( l ) and organoids ( m ) infected for 36 h and 5 d, respectively, with Ctr and immunolabelled for Ki67, γH2AX, and Chlamydia MOMP. l , m Data represent three biological replicates. Source data are provided as a Source Data file.

Article Snippet: Primary cells were expanded in collagen-coated (Sigma, #C3867) tissue culture flasks until they reached 70–80% confluence in human ectocervical primary cell medium (Advanced DMEM/F-12 (Invitrogen, 12634) supplemented with 12 mM HEPES, 1% GlutaMax, 1% B27, 1% N2, 10 ng/mL human EGF (Invitrogen, 15630-056, 35050-038, 17504-044, 17502048, PHG0311), 0.5 μg/mL hydrocortisone (Sigma, H0888-1G), 100 ng/mL human noggin (Peprotech, 120-10 C), 100 ng/mL human FGF-10 (Peprotech, 100-26-25), 1.25 mM N-acetyl-L-cysteine (Sigma, A9165-5G), 2 μM TGF-β receptor kinase Inhibitor IV (SB431542, Calbiochem), 10 μM ROCK inhibitor (Y-27632) (Sigma, Y0503), 10 mM nicotinamide (Sigma, N0636), 10 μM forskolin (Sigma, F6886) before seeding ~20,000 cells/50 μL in Matrigel (BD, #356231) for culturing organoids or for maintenance of 2D stem cells on lethally irradiated J2-3T3 fibroblast feeder cells.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Cell Differentiation