human foreskin fibroblasts hff  (ATCC)

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Evaluation of the biological potential of BM‐MSC/TERT292‐derived EVs enriched from different collection media. (A) Evaluation of the dose‐dependency of the anti‐inflammatory activity of BM‐MSC/TERT292 EVs enriched from different collection media on NO secretion by LPS‐stimulated BV‐2 cells. LPS‐stimulated BV‐2 cells were treated with increasing EV concentrations ranging from 1E+08 to 2E+09 p/mL. 2.5 µM Dexamethasone (Dexa, green dotted line) was included as positive control. Data are represented as mean ± SD and data were normalised on the LPS‐stimulated BV‐2 cells (set at 100%, red dotted line). (B) Assessment of the anti‐fibrosis effect of BM‐MSC/TERT292 EVs enriched from different collection media on α‐SMA induction by TGF‐β1‐stimulated fHDF/TERT166 cells. 20 mM HEPES was included as negative control and 2 µM PP2 was included as positive control. TGF‐β1‐stimulated fHDF/TERT166 cells were treated with a fixed EV concentration of 1E+09 p/mL. (C) Quantification of the wound closure of HUVEC/TERT2 cells upon BM‐MSC/TERT292‐derived EVs treatment. Wound closure was measured at 0, 16 and 24 h after the removal of culture insert and treatment. 20 mM HEPES was included as negative control and complete expansion medium (Medium + FBS) was included as positive control. HUVEC/TERT2 cells were treated with a fixed EV concentration of 1E+09 p/mL. Data are represented as mean ± SD. One‐way Anova was used for the anti‐inflammatory and anti‐fibrosis assay; data were normalised on the LPS‐stimulated BV‐2 cells and TGF‐β1‐stimulated fHDF/TERT166 cells respectively (set at 100%). Two‐way Anova was used for the Wound Healing assay. Significance is shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 4 technical replicates for the anti‐fibrosis and anti‐inflammatory assays, n = 3 biological replicates for the wound‐healing assay). (D) Representative microscope images of the HUVEC/TERT2 cells' wound closure 24 h following culture insert removal and treatment (photos were taken at 100× lens magnification).

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Derivative Assay, Activity Assay, Positive Control, Negative Control, Concentration Assay, Wound Healing Assay, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Evaluation of the biological activity of UCM controls. (A) Evaluation of the anti‐inflammatory activity of the different UCM controls on NO secretion by LPS‐stimulated BV‐2 cells. 20 mM HEPES was included as negative control and 2.5 µM Dexamethasone (Dexa) was included as positive control. LPS‐stimulated BV‐2 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). (B) Assessment of the anti‐fibrosis effect of the different UCM controls on α‐SMA induction by TGF‐β1‐stimulated fHDF/TERT166 cells. 20 mM HEPES was included as negative control and 2 µM PP2 was included as positive control. TGF‐β1‐stimulated fHDF/TERT166 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). (C) Quantification of the wound closure of HUVEC/TERT2 cells upon TUCM control treatment. Wound closure was measured at 0, 16 and 24 h after the removal of culture insert and treatment. 20 mM HEPES was included as negative control and complete expansion medium (Medium + FBS) was included as positive control. HUVEC/TERT2 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). Data are represented as mean ± SD. One‐way Anova was used for the anti‐inflammatory and anti‐fibrosis assay; data were normalised on the LPS‐stimulated BV‐2 cells and TGF‐β1‐stimulated fHDF/TERT166 cells respectively (set at 100%). Two‐way Anova was used for the Wound Healing assay. Significance is shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 4 technical replicates for the anti‐fibrosis and anti‐inflammatory assays, n = 3 biological replicates for the wound‐healing assay). (D) Representative microscope images of the HUVEC/TERT2 cells' wound closure 24 h following culture insert removal and treatment (photos were taken at 100× lens magnification).

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Activity Assay, Negative Control, Positive Control, Derivative Assay, Control, Wound Healing Assay, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Effect of BM‐MSC/TERT292‐derived EVs on fHDF/TERT166 proliferation and metabolism upon enrichment in different collection media. (A) Alamar Blue measurement in fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with BM‐MSC/TERT292‐derived EVs enriched from different collection media. Alamar Blue values were expressed as a percentage of the mean signal from untreated cells (Starvation Medium, set at 100%). (B) Automatic cell count measurement of fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with BM‐MSC/TERT292‐derived EVs enriched from different collection media. fHDF/TERT166 cells were treated with a fixed EV concentration of 1E+09 p/mL. Data are represented as mean ± SD. One‐way Anova was used, and significance is shown as ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 4 technical replicates). (C) Representative microscopy images of Vybrant dye cycle violet stained fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with BM‐MSC/TERT292‐derived EVs enriched from different collection media. fHDF/TERT166 cells were treated with a fixed EV concentration of 1E+09 p/mL. Scale bars: 250 µm.

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Derivative Assay, Cell Characterization, Concentration Assay, Microscopy, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Effect of UCM controls on fHDF/TERT166 proliferation and metabolism. (A) Alamar Blue measurement in fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with different UCM controls. Alamar Blue values were expressed as a percentage of the mean signal from untreated cells (Starvation Medium, set at 100%). (B) Automatic cell count measurement of fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with different UCM controls. fHDF/TERT166 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). Data are represented as mean ± SD; One‐way Anova was used, and significance is shown as ** p < 0.01, **** p < 0.0001 ( n = 4 technical replicates). (C) Representative microscopy images of Vybrant dye cycle violet stained fHDF/TERT166 cells upon 72 h treatment in Starvation Medium with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). Scale bars: 250 µm.

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Cell Characterization, Derivative Assay, Microscopy, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Effect of BM‐MSC/TERT292‐derived EVs on ROS production and tube formation upon enrichment in different collection media. (A) H2DCFDA measurement in BM‐MSC/TERT292 EVs pre‐treated fHDF/TERT166 cells upon H 2 O 2 —mediated oxidative stress induction. H2DCFDA values were expressed as a percentage of the mean signal from H 2 O 2 only—treated cells (Hepes + H 2 O 2 , set at 100%). A fixed concentration of 5% 20 mM Hepes was kept constant in all wells during the BM‐MSC/TERT292‐derived EVs pre‐treatment. fHDF/TERT166 cells were treated with fixed EV concentrations of 2.5 E+08 and 1E+09 p/mL. Data are represented as mean ± SD. One‐way Anova was used, and significance is shown as ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 4 technical replicates). (B) Evaluation of the tube‐formation ability by HUVEC/TERT2 cells terms of number of segments, (C) number of nodes and (D) total segment length upon BM‐MSC/TERT292‐derived EVs treatment. HUVEC/TERT2 cells were treated with a fixed concentration of 1E+09 p/mL. Data are represented as mean ± SD. One‐way Anova was used, and significance is shown as ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 3 technical replicates). (E) Representative microscopy images of tube formation and image analysis by the Angiogenesis Analyzer plug in of ImageJ in HUVEC/TERT2 cells upon 6 h treatment with BM‐MSC/TERT292‐derived EVs enriched from different collection media. Nodes are shown as red dots surrounded by a blue circle, branches are coloured in blue, branches in green, master segments in yellow and mashes in light blue.

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Derivative Assay, Concentration Assay, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT‐Immortalised Mesenchymal Stromal Cells

doi: 10.1002/jev2.70298

Figure Lengend Snippet: Effect of UCM controls on ROS production and tube formation. (A) H2DCFDA measurement in UCM pre‐treated fHDF/TERT166 cells upon H 2 O 2 —mediated oxidative stress induction. H2DCFDA values were expressed as a percentage of the mean signal from H 2 O 2 only—treated cells (Hepes + H 2 O 2 , set at 100%). A fixed concentration of 5% 20 mM Hepes was kept constant in all wells during the UCM pre‐treatment. fHDF/TERT166 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV at 1.0E+09 p/mL concentration enriched in the respective medium (adjusted for the enrichment factor). Data are represented as mean ± SD. One‐way Anova was used, and significance is shown as ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 4 technical replicates). (B) Evaluation of the tube‐formation ability by HUVEC/TERT2 cells terms of number of segments, (C) number of nodes and (D) total segment length upon UCM controls treatment. HUVEC/TERT2 cells were treated with the same UCM volume of the BM‐MSC/TERT292‐derived EV enriched in the respective medium (adjusted for the enrichment factor). Data are represented as mean ± SD. One‐way Anova was used, and significance is shown as ** p < 0.01, *** p < 0.001, **** p < 0.0001 ( n = 3 technical replicates). (E) Representative microscopy images of tube formation and image analysis by the Angiogenesis Analyzer plug in of ImageJ in HUVEC/TERT2cells upon 6 h treatment with different UCM controls. Nodes are shown as red dots surrounded by a blue circle, branches are coloured in blue, branches in green, master segments in yellow and mashes in light blue.

Article Snippet: Human foreskin fibroblasts fHDF/TERT166 (Evercyte GmbH) were cultured in DMEM/F12 (PAN Biotech), supplemented with 10% FBS (PAN Biotech) and 100 μg/mL G418 (InvivoGen).

Techniques: Concentration Assay, Derivative Assay, Microscopy

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: (A) BF images five out of the seven cell lines, including telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive line VA13. (B) Quantification of hTAPAS and hTERT mRNA levels by qRT-PCR. (C) RT–PCR analysis of hTERT splice isoforms using primers spanning exons 2–3 and 5–9. An inverse relationship between hTAPAS and hTERT expression was observed in HFF-1 and BJ fibroblasts (low hTERT , detectable hTAPAS ) and in HEK293T and NALM6 cells (high hTERT , absent hTAPAS ), whereas intermediate patterns were detected in IMR90, VA13, and iPSCs, with iPSCs maintaining moderate hTERT expression despite high hTAPAS levels.

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: Targeted DNA methylation profiling was performed using CRISPR–Cas9 enrichment followed by Nanopore sequencing across a ∼9 kb region spanning hTAPAS through hTERT intron 2 (Chr. 5: 1,196,006–1,205,206) and a ∼6.5 kb region covering introns 6–8 (Chr. 5: 1,174,035–1,180,535). The analysis included telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive cell lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive cell line VA13. DNA methylation levels at individual CpG sites are depicted across the indicated genomic regions, including hTAPAS , the THOR region, the core promoter, exon1, intron1, exon 2 and intron 2. Methylation for each individual CpG is shown as a percentage, with unmethylated CpGs depicted in red and methylated CpGs in blue. Methylation across intron 2 was consistently high (80–100%) in all cell lines, whereas regions encompassing hTAPAS , the THOR region, exon 2, and introns 6–8 displayed marked variability between cell types. CpGs within the hTAPAS region were highly methylated in telomerase-positive cells and in the ALT-positive VA13 line, but largely unmethylated in fibroblasts, with partial methylation observed in IMR90. The core hTERT promoter and exon 2–proximal regions remained mostly unmethylated in all cell lines except VA13, which exhibited substantial hypermethylation

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: DNA Methylation Assay, CRISPR, Nanopore Sequencing, Methylation

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: (A) BF images five out of the seven cell lines, including telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive line VA13. (B) Quantification of hTAPAS and hTERT mRNA levels by qRT-PCR. (C) RT–PCR analysis of hTERT splice isoforms using primers spanning exons 2–3 and 5–9. An inverse relationship between hTAPAS and hTERT expression was observed in HFF-1 and BJ fibroblasts (low hTERT , detectable hTAPAS ) and in HEK293T and NALM6 cells (high hTERT , absent hTAPAS ), whereas intermediate patterns were detected in IMR90, VA13, and iPSCs, with iPSCs maintaining moderate hTERT expression despite high hTAPAS levels.

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: Targeted DNA methylation profiling was performed using CRISPR–Cas9 enrichment followed by Nanopore sequencing across a ∼9 kb region spanning hTAPAS through hTERT intron 2 (Chr. 5: 1,196,006–1,205,206) and a ∼6.5 kb region covering introns 6–8 (Chr. 5: 1,174,035–1,180,535). The analysis included telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive cell lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive cell line VA13. DNA methylation levels at individual CpG sites are depicted across the indicated genomic regions, including hTAPAS , the THOR region, the core promoter, exon1, intron1, exon 2 and intron 2. Methylation for each individual CpG is shown as a percentage, with unmethylated CpGs depicted in red and methylated CpGs in blue. Methylation across intron 2 was consistently high (80–100%) in all cell lines, whereas regions encompassing hTAPAS , the THOR region, exon 2, and introns 6–8 displayed marked variability between cell types. CpGs within the hTAPAS region were highly methylated in telomerase-positive cells and in the ALT-positive VA13 line, but largely unmethylated in fibroblasts, with partial methylation observed in IMR90. The core hTERT promoter and exon 2–proximal regions remained mostly unmethylated in all cell lines except VA13, which exhibited substantial hypermethylation

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: DNA Methylation Assay, CRISPR, Nanopore Sequencing, Methylation