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: To maintain bud tip progenitor organoids in a progenitor state, serum-free basal medium was further supplemented with FGF7 (10 ng/mL, Recombinant Human Fibroblast Growth Factor 7; R&D Systems cat. no. 251-KG/CF), CHIR99021 (3 μM, Stem Cell Technologies cat. no. 72054), and All Trans Retinoic Acid (ATRA; 50 nM, Stemgent cat. no. 04-0021, CAS Number 302-79-4).

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

Journal: Stem Cell Research & Therapy

Article Title: Bronchioalveolar stem cells derived from mouse-induced pluripotent stem cells promote airway epithelium regeneration

doi: 10.1186/s13287-020-01946-7

Figure Lengend Snippet: Differentiation of iPSCs into BASCs. a Schema of iPSC differentiation procedure: iPSCs were differentiated for 24 days through hanging-drop-based formation of embryoid bodies (EBs); the BM was supplemented from 0 to 24 days with 20 ng/mL keratinocyte growth factor (KGF) and DCI (① d10–d24 or ② d14–d24). EBs were induced using the hanging-drop method for the first 3 days, and the obtained EBs were transferred at 3 days to super-low-adherent culture dishes and then at 5 days to adherent culture dishes. Cells were cultured until 24 days in the medium. b Pluripotency of undifferentiated iPSCs (0 days) at passage 25. Immunofluorescence labelling of mouse iPSCs for the stem cell markers OCT4, SOX2, and SSEA-1. The GFP gene was knocked-in under the Nanog promoter, which allowed detection of GFP (green) in undifferentiated cells. Scale bar = 100 μm. c Flow cytometry analysis for BASC identification. Comparison of protocols ① d10–d24 DCI and ② d14–d24 DCI revealed that BASC differentiation efficiency did not differ significantly between the protocols ( P > 0.05, Student’s t test); horizontal line inside indicates median and whiskers indicate min to max values. DCI, 10 nM dexamethasone plus 0.1 mM 8-bromoadenosine 3′5′-cyclic monophosphate sodium salt and 0.1 mM 3-isobutyl-1-methylxanthine; iPSCs, induced pluripotent stem cells; BASCs, bronchioalveolar stem cells; GFP, green fluorescent protein

Article Snippet: The BM was further supplemented with the following growth factors: 20 ng/mL recombinant human keratinocyte growth factor (KGF; ProteinTech Inc., Tokyo, Japan) (from 0 to 24 days) and DCI (treatment protocol: 1 d10–d24 or 2 d14–d24).

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Comparison

Journal:

Article Title: Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas

doi:

Figure Lengend Snippet: Expression of FGF-1, FGF-7, and FGF-10 and their receptor FGFR2b during pancreas development. (A) Reverse transcriptase–PCR was carried out on pancreatic cDNAs from the indicated stages of development by using appropriate primers for each FGF. Thirty-five cycles of amplification were used for the amplification of FGFs transcripts. Cyclophilin amplification (30 cycles) was used to control the quality of the cDNAs. (B) Expression of FGFR2b mRNA in the developing rat pancreas. FGFR2b expression was analyzed by Northern blot. Total RNA from pancreases at different stages of development were hybridized successively with FGFR2b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes.

Article Snippet: The culture medium consisted of RPMI medium 1640 without serum and supplemented or not with 50 ng/ml of either recombinant human FGF-1 (Boehringer Mannheim), recombinant human FGF-7 (Becton Dickinson), or recombinant human FGF-10.

Techniques: Expressing, Amplification, Northern Blot

Journal:

Article Title: Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas

doi:

Figure Lengend Snippet: Mitogenic and morphogenic effects of FGF-1, FGF-7, and FGF-10. (A) DNA content of E11.5 mesenchyme-free pancreatic epithelia cultured during 7 days in the absence or presence of FGF-1, FGF-7, or FGF-10 (50 ng/ml). Each data point represents the mean ± SEM of 10 pancreatic rudiments. (B-D) Effects of FGF-1 and FGF-7 on pancreatic morphogenesis in Matrigel (B) absence of exogenous FGF, (C) FGF-1, 50 ng/ml, and (D) FGF-7, 50 ng/ml.

Article Snippet: The culture medium consisted of RPMI medium 1640 without serum and supplemented or not with 50 ng/ml of either recombinant human FGF-1 (Boehringer Mannheim), recombinant human FGF-7 (Becton Dickinson), or recombinant human FGF-10.

Techniques: Cell Culture

Journal:

Article Title: Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas

doi:

Figure Lengend Snippet: Immunohistological analysis of pancreatic epithelium grown in vitro in the presence of FGFs. E11.5 mesenchyme-free pancreatic epithelia were grown in collagen gels for 7 days in the absence (A and B) or presence of 50 ng/ml of either FGF-1 (C and D), FGF-7 (E and F), or FGF-10 (G and H). The development of the endocrine tissue (A, C, E, and G) was evaluated after anti-glucagon (red fluorescence) and anti-insulin (green fluorescence) staining. The development of the exocrine tissue (B, D, F, and H) was evaluated after anti-amylase (green fluorescence) staining. The effect of FGF-1 and FGF-7 on acinar cell proliferation (B, D, and F) was analyzed by staining with the antimitotic proteins, mpm2 antibody (red fluorescence). Magnifications: A and B, ×600; C-H, ×400.

Article Snippet: The culture medium consisted of RPMI medium 1640 without serum and supplemented or not with 50 ng/ml of either recombinant human FGF-1 (Boehringer Mannheim), recombinant human FGF-7 (Becton Dickinson), or recombinant human FGF-10.

Techniques: In Vitro, Fluorescence, Staining