Journal: Advanced Healthcare Materials

Article Title: Generation of an Induced Pluripotent Stem Cell‐Derived Alveolar Type II In Vitro Model to Study Influenza A Virus Infection and Drug Treatments

doi: 10.1002/adhm.202405141

Figure Lengend Snippet: Differentiation, cultivation, and characterization of iAT2 cells. (a) Overview of the differentiation, cultivation, and plating procedure of iAT2s as described in the methods. iPSCs: induced pluripotent stem cells; iAT2s: iPSC‐derived alveolar type II cells; ALI: air–liquid interface; NKX2.1: NK2 homeobox 1; SP‐C: surface active agent protein C; for StemDiff, DS/SB, CBRa, CK/DCI, CK/DCI+Y: see methods. (b) Representative flow cytometry plots of the lung progenitor markers NKX2.1 coupled to GFP on day 14 (left) and SP‐C coupled to tdTomato on day 30 (right). (c) Representative transmission light image of alveolospheres within matrigel before passaging on day 35. Scale bar = 30 µm. (d) Representative transmission light images of iAT2s seeded on transwell inserts directly after seeding (day X) and 4 or 6 days after seeding (day X + 4 and X + 6). Scale bars = 50 µm. (e) Immunofluorescence images of iAT2s showing expression of antigen Kiel 67 (Ki67; green, left), SP‐C (magenta, left), lysophosphatidylcholine acyltransferase 1 (LPCAT1; yellow, middle), and NKX2.1 (green, right). Nuclei are displayed in blue. Single z‐planes are shown. Scale bars = 20 µm (left, middle) or 50 µm (right).

Article Snippet: Transwell inserts (Greiner Bio‐one, 662 641) were placed in 24‐well plates and coated with 100 μL of Corning Matrigel Human Embryonic Stem Cell‐qualified matrix (Corning, 354 277) according to the manufacturer's instructions.

Techniques: Derivative Assay, Flow Cytometry, Transmission Assay, Passaging, Immunofluorescence, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Vimentin staining on immunohistochemistry used to identify and quantify fibroblasts in the ureter. The healthy ureter adjacent to the treatment site (A–D and I) and the irreversible electroporation (IRE)-treated ureter (E–H and J) were collected at 1, 7, 14, and 28 days after treatment. An increased presence of fibroblasts (arrow) was observed in the healthy tissue adjacent to the treatment site only in day 1 samples (A and I). In comparison, the peak population of fibroblasts was observed at day 7 in the IRE-treated ureter (F and J) and remained greater than that of the surrounding healthy tissue (B–D and I) at all subsequent time points (G, H, and J). Other than fibroblasts (arrow), vimentin staining was also observed in the blood vessel wall within the treated ureter in day 28 samples (H, arrowhead) and was censored from measurements. Samples were collected from two treatment sites in two animals at each indicated time point. Scale bar = 50 μm. *P < 0.05; **P < 0.01. Staining levels in untreated control samples are denoted with a dashed line.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Immunohistochemistry, Electroporation, Comparison, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: α-Smooth muscle actin (α-SMA) staining with immunohistochemistry used to identify and quantify myofibroblasts in the ureter. The healthy ureter adjacent to the treatment site (A–D and I) and the irreversible electroporation (IRE)-treated ureter (E–H and J) were collected at 1, 7, 14, and 28 days after treatment. The myofibroblast population (F and G, arrow) in the healthy ureter surrounding the treatment site appeared similar at all time points (A–D and I), whereas the population peaked at day 7 (F and J) in the IRE-treated ureter and remained greater than the surrounding healthy tissue (B–D and I) at all subsequent time points (G, H, and J). Apart from fibroblasts, α-SMA staining was also observed in smooth muscle cells found in blood vessels and regenerating smooth muscle in the treated ureter in day 28 samples (H, arrowhead) and was censored from measurements. Samples were collected from two treatment sites in two animals at each indicated time point. Scale bar = 50 μm. **P < 0.01. Staining levels in untreated control samples are denoted with a dashed line.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Immunohistochemistry, Electroporation, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Evaluation of macrophage chemoattractant and proliferation in the irreversible electroporation (IRE)-treated ureter. The nuclei of cells in the IRE-treated ureter wall (brown; A) stained strongly positive for TUNEL at day 1 posttreatment, suggesting ongoing cell death. Arrows indicate the ureteral lumen and basal membrane, with the notable absence of the urothelium, which is expected to slough as a consequence of IRE. B−E: multiplex immunofluorescence with DAPI (blue; B) for the nucleus, Iba-1 (red; C) for macrophages, Ki-67 (green; D) for cell proliferation, and merged image (E) showing the collocation of Ki-67 and Iba-1 staining (arrow), suggesting the presence of proliferating macrophages in the ureteral wall at day 7 post-IRE.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Electroporation, Staining, TUNEL Assay, Membrane, Multiplex Assay, Immunofluorescence

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Iba-1 staining on immunohistochemistry used to identify macrophages in the ureter. The healthy ureter adjacent to the treatment site (A–D and I) and the irreversible electroporation (IRE)-treated ureter (E–H and J) were collected at 1, 7, 14, and 28 days after treatment. The macrophage population in the healthy ureter surrounding the treatment site was briefly elevated in day 1 samples (A and I) but reverted to reduced levels at subsequent time points (B–D and I). The peak macrophage population in the IRE-treated ureter (G and H, arrow) was observed at day 7 (F and J) and remained greater than the surrounding healthy tissue (B–D and I) at all subsequent time points (G, H, and J). Samples were collected from two treatment sites in two animals at each indicated time point. Scale bar = 50 μm. **P < 0.01. Staining levels in untreated control samples are denoted with a dashed line.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Immunohistochemistry, Electroporation, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Masson’s trichrome staining for collagen for the quantification of ureteral wall scarring after irreversible electroporation (IRE). The IRE-treated ureter showed a progressive loss of normal ureteral wall architecture with increased collagen content, with a steady increase in levels on day 1 (A) compared with subsequent time points at day 7 (B), day 14 (C), and day 28 (D). Sparse regenerating blood vessels and smooth muscle (arrow) can be seen at day 28 (D), but the overall ureteral wall architecture was lost to scarring. E: percentage of the positive area at the indicated time points. See Supplemental Fig. S1 (https://doi.org/10.5281/zenodo.2538189) for comparative images of collagen staining in the healthy ureteral wall adjacent to the treatment site. Samples were collected from two treatment sites in two animals at each indicated time point. Scale bar = 200 μm. **P < 0.01. Staining levels in untreated control samples are denoted with a dashed line.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Electroporation, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Transforming growth factor (TGF)-β1 staining and quantification of expression in fibroblasts (spindle-shaped cells) in the irreversible electroporation (IRE)-treated ureter. A−D: TGF-β1-positive cells (arrow) in the swine ureter treated with IRE at day 1 (A) day 7 (B), day 14 (C), and day 28 (D). E: quantification of immunohistochemical staining indicating that the population of fibroblasts staining positive for TGF-β1 peaked at day 7 (B and E), with a gradual decrease at subsequent time points (C–E). See Supplemental Fig S1 (https://doi.org/10.5281/zenodo.2538189) for comparative images of TGF-β1-positive cells in the healthy ureteral wall adjacent to the treatment site. Samples were collected from two treatment sites in two animals at each indicated time point (n = 4). Scale bar = 50 μm. **P < 0.01. Staining levels in untreated control samples are denoted with a dashed line.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Expressing, Electroporation, Immunohistochemical staining, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Macrophage (top) and transforming growth factor (TGF)-β1 (bottom) staining in the irreversible electroporation (IRE)-treated ureter at days 1, 7, 14, and 28 from matched tissue sections. Comparison of macrophage (Iba-1; A–D) and (TGF-β1; E–H) immunohistochemical stains suggested TGF-β1 staining in macrophages (arrows) on days 1–14, and staining could also be observed in vascular cells at day 14 and 28 (arrowheads). Scale bar = 100 μm. Samples were collected from two treatment sites in two animals at each indicated time point.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Staining, Electroporation, Comparison, Immunohistochemical staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Transforming growth factor (TGF)-β1 secretion by macrophages and fibroblasts in vitro. Macrophages were incubated with vehicle (A), LPS (B), irreversible electroporation (IRE)-treated RAW 264.7 cells (C), or IRE-treated 3T3 fibroblasts (D). All treatments resulted in activation as observed by a morphological dendritic shape (white arrows, B–D). RAW 264.7 macrophages demonstrated increased secretion of TGF-β1 upon activation with IRE-treated 3T3 cells killed with IRE (E). Compared with control or IRE treatment, 3T3 cells stimulated with TGF-β1 after IRE demonstrated increased autocrine secretion of TGF-β1 at 48 h post-IRE (F). IRE treatment reduced the population and proliferation of 3T3 cells in vitro, the effects of which were not overcome with recombinant human (h)TGF-β1 stimulation (G). *P < 0.05; **P < 0.02.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: In Vitro, Incubation, Electroporation, Activation Assay, Control, Recombinant

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Effect of transforming growth factor (TGF)-β1, irreversible electroporation (IRE), or TGF-β1 + IRE on α-smooth muscle actin (α-SMA) synthesis in 3T3 fibroblasts. Compared with control or IRE treatment alone, stimulation of 3T3 cells with recombinant human (h)TGF-β1 was necessary and sufficient to promote α-SMA synthesis as early as 24 h post-IRE (A), the effects of which persisted through 48 h post-IRE (B). Treatment with pirfenidone (PFD) attenuated α-SMA at all time points. *P < 0.01; **P < 0.001.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Electroporation, Control, Recombinant

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Effect of transforming growth factor (TGF)-β1 stimulation on collagen production, profibrotic mRNA expression in 3T3 fibroblasts, and TGF-β1 production in macrophages in combination with irreversible electroporation (IRE) and pirfenidone (PFD). Treatment with PFD reduced TGF-β1 secretion by RAW 264.7 macrophages activated with IRE-treated 3T3 cells (A). In 3T3 cells stimulated with recombinant human (h)TGF-β1, there was increased collagen production (B) and mRNA expression of both α-smooth muscle actin (ACTA2) and collagen [collagen type I-α1 (COL1A1); C]. Both collagen production and mRNA expression were reduced with PFD treatment. *P < 0.05; **P < 0.01.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Expressing, Electroporation, Recombinant

Journal: American Journal of Physiology - Renal Physiology

Article Title: Macrophage-secreted TGF-β 1 contributes to fibroblast activation and ureteral stricture after ablation injury

doi: 10.1152/ajprenal.00260.2018

Figure Lengend Snippet: Effect of pirfenidone (PFD) and irreversible electroporation (IRE) treatment of macrophages and fibroblasts. Fibroblasts (A) and macrophages (B) demonstrated increased annexin V expression after treatment with IRE, PFD, or a combination of the two. Stimulation with transforming growth factor (TGF)-β1 somewhat reduced the number of apoptotic cells after IRE, but this effect was overcome with the addition of PFD to the treatment regimen. *P < 0.02; **P < 0.01.

Article Snippet: In vitro IRE was performed on cells in 24-well plates using two cylindrical electrodes (25-mm length, 0.8-mm diameter, and 10-mm spacing between electrodes) suspended into the well using three-dimensional printed electrode holders or using electroporation cuvettes having plate electrodes at 4-mm gap (Bio-Rad).

Techniques: Electroporation, Expressing