Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Early epidermal cell fate determination under the influence of multiple signaling pathways in hESCs. See also . A. Stagewise method for keratinocyte differentiation. B. The time course of epidermal differentiation under TGFβ inhibition. hESCs were treated with SB431542 (SB) in differentiation medium (See Materials and Methods), and the cells were harvested at specific time points for analysis of gene expression by RT-qPCR. The results were normalized to GAPDH. C. The emergence of TP63 under pulse treatment of TGFβ inhibitor. hESCs were treated with SB431542 (SB) for specified periods in differentiation medium followed by differentiation medium only until day 6. Cells were collected on day 6 for analysis of gene expression by RT-qPCR. The results were normalized to GAPDH. D. BMP4 promotes TP63 expression under TGF-β inhibition. hESCs were treated with SB431542 (SB) in differentiation medium, and BMP4 was added from day 1 till day 6 when cells were harvested for gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. E. The impact of WNT pathway modulation on epidermal cell fate determination. hESCs were treated with SB431542 (D0-6) and BMP4 (D1-6) in differentiation medium, and CHIR99021(CHIR, 5 µM) or IWR-1 (1 μM) was added from day 1 to day 6. Cells were then harvested for gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. ns, not significant.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Protein-Protein interactions, Inhibition, Gene Expression, Quantitative RT-PCR, Expressing

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Optimization of epidermal cell fate determination at early stage. See also . A. The time course of primitive ectoderm induction. Cells were treated with SB431542 single treatment for 0, 1, 2 or 3 days in differentiation medium, followed by combination treatment with SB431542, BMP4 and CHIR99021 until day 6, and collected on day 6 for TP63 gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. B. The impact of prolonged exposure to TGFβ inhibitor. H1 cells were treated with TGFβ inhibitor SB431542 (SB) for 2, 4 or 7 days in the presence of BMP4 (Day 1 to 7) and CHIR99021 (CHIR, Day 1 to 5) in differentiation medium, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no SB4315432 treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. C. The effect of BMP4 timing on TP63 induction. In the presence of SB431542 (SB, Day 0-6) and CHIR99021 (CHIR, Day 1-5), BMP4 was added for specific time periods, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no BMP4 treatment); Data are presented as the mean±SD of three independent experiments. ns, not significant (one-way ANOVA with post hoc multiple comparisons). D. The effect of WNT activation timing on epidermal differentiation. WNT activator CHIR99021 (CHIR) was added in the presence of SB431542 (SB, Day 0-6) and BMP4 (Day 1-7) for specified periods of time, and gene expression was analyzed by RT-qPCR on day 7. The results were normalized to control (no CHIR treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. E. Impact of NOTCH inhibition during epidermal differentiation. Epidermal differentiation was carried out with SB431542 (SB, Day 0-6), CHIR99021 (CHIR, Day 1-6) and BMP4 (Day 1-6). NOTCH inhibitor DAPT was added between Day 4-6, and the gene expression was analyzed by qPCR on Day 6. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. F. The impact of WNT activation along with NOTCH inhibition on epidermal cell fate determination. Cells in epidermal differentiation were treated with DAPT with or without CHIR99021 (CHIR) between day 4 to 6, and the samples were collected on Day 6 for RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Control, Activation Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Optimization of keratinocyte maturation conditions. See also . A. BMP4 and NOTCH inhibitor (DAPT) treatments are sufficient to support keratinocyte maturation from day 6 to day 8. Beyond Day 6 of differentiation, cells were treated with or without SB431542 (SB) or CHIR99021 (CHIR) under BMP4 and NOTCH inhibitor treatment for two extra days, and the gene expression was examined by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. B. Immunostaining of TP63 expression on day 0 and day 8. Scale bar, 50 µm. C. The impact of calcium concentration on the expression of keratinocyte markers. After eight days of differentiation, cells were maintained in different calcium concentrations (1 mM versus 0.06 mM) between day 9 and day 11, and analyzed for gene expression. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. D. Immunostaining of TP63 (green) and KRT14 (red) expression on D8 and D11. Scale bar, 50 µm. White arrow, KRT14 expression. E. Flow cytometry analysis of TP63 on Day 8 and Day 11 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Quantification of TP63-positive cells on Day 0, Day 8 and Day 11 by flow cytometric analysis. Data shown are mean±SD of three independent experiments. *, p < 0.05.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Immunostaining, Expressing, Concentration Assay, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Keratinocyte derivation procedure in defined conditions. See also . A. Schematic drawing of the optimized keratinocyte differentiation protocol. B. Cell morphology changes in the differentiation process. Scale bar,100µm. C. Stagewise keratinocyte gene expression by RT-qPCR. The results were normalized to hESC (D0); Data are representative of 3 independent experiments. D. Immunostaining of TP63 (red), KRT14 (green), KRT1 (green) and KRT10 (green) on day 24 and day 32 of differentiation. Scale bar, 50µm. E. Flow cytometry analysis of KRT14 (left panel) and KRT1 (right panel) on Day 28 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Keratinocyte differentiation from multiple hESC (H1, H9) and hiPSC (ND1-4, NL-1, NL-4) lines. Gene expression was analyzed on day 20 of differentiation and compared with HaCaT keratinocyte cell line, primary human foreskin keratinocytes (HFK-1, HFK-2) and undifferentiated hESCs (H1). The results were normalized to hESC (H1); Data are representative of 3 independent experiments.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

doi: 10.3390/ijms22179114

Figure Lengend Snippet: Cell surface expression of immune stimulatory checkpoint molecules CD70 ( a ), CD137-L ( b ) and ICOS-L ( c ) on the cell surface of HPV-negative (grey dots) and HPV-positive (white dots) HNSCC cell lines with n hpv− = 5; n hpv+ = 6 biological independent experiments. Cells were subjected to either a hypofractionated irradiation regimen (5x3.0Gy) or a single high dose of 19.3Gy. ΔMFI was calculated by subtracting the respective stained samples from Zombie-only-stained samples. Graphs show Median + min to max and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05.

Article Snippet: ICOS-L (CD 275) , BV 421 , BD Bioscience , 564,276.

Techniques: Expressing, Irradiation, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

doi: 10.3390/ijms22179114

Figure Lengend Snippet: Normalized gene expression of immune suppressive (PD-L1 ( a ), PD-L2 ( b ), HVEM ( c ) and immune stimulatory (CD70 ( d ), CD137-L ( e ), ICOS-L ( f )) checkpoint molecules of HPV-negative (grey dots) and HPV-positive (white dots) HNSCC cell lines. Cells were subjected to either a hypofractionated irradiation regimen (5x3.0Gy) or a single high dose of 19.3Gy. Normalized gene expression was calculated by normalizing samples to the following housekeeping genes: HPV−: ACTB, RPL27, RPL30, RPS18; HPV+: ACTB, RPL27, RPL30, RPS18, UBC. Graphs show Median + min to max for n HSC-4 = 4, n Cal33 = 5, n UD-Scc-2 = 5, n UM-Scc-47 = 3 biological independent experiments and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05.

Article Snippet: ICOS-L (CD 275) , BV 421 , BD Bioscience , 564,276.

Techniques: Expressing, Irradiation, MANN-WHITNEY