Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Schematic of lung epithelial development. As the airways extend, bud tip progenitors are maintained as progenitors in the tips of branching buds, and leave cells behind that give rise to the intrapulmonary airways. Late in development, remaining bud tip progenitors differentiate into alveolar cells. No bud tip progenitors are present in the adult lung. B) Schematic of creation of epithelium-only bud tip progenitor organoids from 12 week fetal lungs. C) Feature plots for bud tip progenitor marker genes SFTPC, ID2 and HMGA1 from scRNA-seq of day 0 bud tip progenitor organoids maintained for 3 weeks in culture. Additional feature plots from this same data set are shown in Figure 4A. D) mRNA expression by QRT-PCR of basal cell marker TP63 in bud tip progenitor organoids treated for 3 days with serum-free basal medium supplemented with DMSO (control) or with signaling factors known to be important for lung development and cellular differentiation. DMSO (1:1,000 dilution), FGF7 (10 ng/mL), ‘3F’ (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), Dexamethasone (25 ng/mL), IL6 (10 ng/mL), IL2 (50 U/mL), Smoothened Agonist (SAG; 500 nM), EGF (100 ng/mL), FGF10 (500 ng/mL), CHIR99021 (2 μM), IFNγ (10 ng/mL), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), DAPT (10 μM), Hydrocortisone (100 ng/mL). Gene expression is reported as arbitrary units. Treatment with TGFβ1 led to a significant increase in the expression of TP63 (one-way Analysis of Variance (ANOVA) (alpha=0.05, p<0.0001, F=14.7. Dunnett’s test of multiple comparison’s compared the mean of each group to the mean of the DMSO control group.) Estimated p values are shown on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. E) Bud tip progenitor organoids were treated with FGF7 (10 ng/mL), a permissive environment for TP63 expression compared to maintenance in bud tip progenitor medium (‘3F’), or with FGF7 (10 ng/mL) plus factors to inhibit SMAD signaling (A8301 [1 μM] and NOGGIN [100 ng/mL]) and TP63 gene expression was evaluated by QRT-PCR after 10 days in culture. A one-way Analysis of Variance was used followed by Tukey’s multiple comparison test to compare the means of each group to the mean of every other group. Estimated p values are reported on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. F) Bud tip progenitor organoids were treated for 3 days with SMAD activation or inhibition conditions and expression of TP63 was evaluated by QRT-PCR for all treatment groups. All media contained 3F components (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), with individual groups containing combinations of: DMSO (1:1000 dilution), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), SB431542 (10 μM), LDN212854 (200 nM). One-way ANOVA alpha=0.05, F=21.19, p<0.0001; Tukey’s multiple comparisons of the mean of each group versus the mean in all other groups, estimated p values are reported on the graph. 3 days TGFβ1 and BMP4 is referred to as ‘dual SMAD activation’, or ‘DSA’. Data is plotted as arbitrary units. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment and is representative of n=3 experiments. G) QRT-PCR for markers of canonical differentiated lung epithelial cell types showing DMSO (gray bars) and DSA treated (blue bars) organoids after 3 total days of treatment. Data is plotted as fold change over DMSO controls. Two-sided Mann-Whitney Tests were performed to compare the mean of the DMSO group to the Dual Smad Activation group (alpha=0.05). Error bars represent the mean +/− the standard error of the mean. n=3 independent biological specimens, and data is from a single experiment and is representative of n=3 experiments. H) Protein staining of DMSO treated (control) fetal bud tip progenitor organoids (top row) and 3 days of DSA treatment (bottom row) for TP63+ protein (green), KRT5 (pink) and DAPI (blue). Scale bar represents 50 μm. I) Quantification of (g). Total number of TP63+ cells were counted for 3–9 individual organoids across 3 biological replicates. n=3 independent biological specimens. A two-sided Mann Whitney test (alpha=0.05) was used to compare the means of each sample. For all graphs, p values are reported as follows: * p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Marker, Expressing, Quantitative RT-PCR, Control, Cell Differentiation, Gene Expression, Comparison, Activation Assay, Inhibition, MANN-WHITNEY, Staining

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Clinical Cancer Research

Article Title: Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12

doi: 10.1158/1078-0432.ccr-13-0943

Figure Lengend Snippet: Figure 1. Characterization of hamster anti-CXCL12 antibodies. A, dose-dependent inhibition of CXCL12a-induced Jurkat cell migration in the absence or presence of various concentrations of hamster antibodies. Of note, 10 ng/mL human CXCL12a was preincubated with antibodies in serial 1:3 dilutions at room temperature for 30 minutes. The highest concentration used for each antibody is shown. IC50 was the average of 3 independent experiments. Data are expressed as mean SD. B, relative potencies of various antibodies in inhibiting 125I-CXCL12a binding to 293-CXCR4 or (C) 293-CXCR7 cells. The same batch of antibodies was used in two binding assays, with AMD3100 as a control. Results shown are representative of 2 independent experiments. The cell-bound 125I-CXCL12a was determined and the results are expressed as percentage versus negative control of no antibody treatment. D, effect of 30D8 or control hamster antibody gD:2566 (Genentech) at 20 mg/mL on freshly isolated bone marrow cell migration induced by mouse KC, MIP2, and RANTES at 20 or 100 ng/mL. Data are expressed as mean SD. E, effects of 30D8 on CXCL12a-induced Jurkat cell Rac activation. Jurkat cells were treated with 100 ng/mL human CXCL12a for 10 minutes in the presence of various concentrations of 30D8, 46H9, 18E9, or control hamster antibody, MAB170 or AMD3100, as indicated. The total and GTP-bound Rac1 were detected by Western blotting using an antibody against Rac1. Data were representative of 3 independent studies.

Article Snippet: Hamster immunization and splenic fusion Female Armenian hamsters were hyperimmunized intraperitoneally, twice per week for a total of 12 to 13 boosts, with 2 mg/animal recombinant human CXCL12a (R&D Systems).

Techniques: Inhibition, Migration, Concentration Assay, Binding Assay, Control, Negative Control, Isolation, Activation Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12

doi: 10.1158/1078-0432.ccr-13-0943

Figure Lengend Snippet: Figure 4. Crystal structure of hu30D8 Fab in complex with human CXCL12a A human CXCL12a dimer is shown as ribbon diagram, green and magenta. Two Fab molecules (shown as surface rendering) and human CXCL12a dimer constitute the crystallographic asymmetric dimer. Blue, heavy chain; orange, light chain. B, a close-up view of the interface. In the front, CXCL12a fragment that engages direct interactions with hu30D8 are shown in green ribbons. In the back, hu30D8 is shown as surface rendering, heavy chain in blue, light chain in orange. The paratope is colored in red. The bright red patches comprise atoms within 4 Å from human CXCL12a; pink patches comprise atoms within 4–4.5 Å. C, the specific interactions around the "hot spot" N44 and N45 of human CXCL12a. Color-coding for the carbon atoms: green, human CXCL12a; blue, Fab heavy chain; orange, Fab light chain. Other atoms are colored by type: blue, nitrogen; red, oxygen. D, the Fab interaction involving RFFESH fragment of human CXCL12a. Color-coding is the same as in C.

Article Snippet: Hamster immunization and splenic fusion Female Armenian hamsters were hyperimmunized intraperitoneally, twice per week for a total of 12 to 13 boosts, with 2 mg/animal recombinant human CXCL12a (R&D Systems).

Techniques: