Progres c3 camera

The chemotactic capacity of EVs, CM and EV-depleted CM of DPSCs and BM-MSCs on endothelial cells was tested in a Boyden chamber migration assay. HUVECs were seeded at a density of 3,500 cells/mm² in serum-free low-glucose DMEM medium in the inserts of a HTS Transwell-96 permeable support with pores of 8 µm (Corning). Different concentrations of CM, EV-depleted CM (25X, 5X, 1X, 1/5, 1/25) and EVs (1X, 1/2, 1/4, 1/8) in DMEM medium of both stem cell types were added to the lower compartment in a volume of 100 µL. Low-glucose DMEM medium with or without 10% FBS in the lower wells was used as positive and negative control, respectively. After 24 h of incubation, transmigrated cells were fixed by 4% PFA and stained with 0.1% crystal violet (Sigma-Aldrich) in 70% ethanol. Representative pictures were taken by a Nikon eclipse TS100 inverted microscope with a Jenoptik ProgRes C3 camera (Jenoptik, Jena, Germany). Migration was quantified as the mean area percentage covered on the lower side of the membrane by Axiovision 4.6 Software (Carl Zeiss Vision, Aalen, Germany).

Colorimetric whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) were performed as elsewhere described [98 (link), 99 (link)]. The following DIG- (Roche), FITC- (Roche), or DNP- (Perkin Elmer) labeled riboprobes were synthesized using an in vitro transcription kit (Roche): Smed-β-catenin1/3/4, Smed-TCF1/2/3; Smed-opsin, Smed-otxA, Smed-sp6/9 [52 (link)]; Smed-tph [100 (link)]; Smed-ovo [54 (link)]; Smed-h2b [101 (link)]; Smed-th (tyrosine hydroxylase), Smed-tbh (tryptophan hydroxylase) [102 (link)], Smed-pc2 (prohormone convertase 2) [103 (link)]. Primers used for their synthesis are indicated (S1 Table). Riboprobes were finally diluted to 250 ng/μL in pre-hybridization solution, stored at -20°C, and were used at 1:500 in hybridization solution, except for sp6/9 (1:200). Samples were observed through Leica MZ16F (Leica Microsystems, Mannhiem, BW, Germany), Zeiss Stemi SV6 stereomicroscopes and a Zeiss Axiophot microscope (Zeiss, Jena, TH, Germany); images were captured with a ProgRes C3 camera from Jenoptik (Jena, TH, Germany), sCMEX 3.0 camera (Euromex, Arnhem, The Netherlands) and Leica DFC300FX camera (Leica Microsystems, Heerbrugg, CH, Switzerland). Confocal laser scanning microscopy was performed with a Leica TCS-SP2 (Leica Lasertchnik, Heidelberg, BW, Germany) adapted for an inverted microscope.

FISH and immunostaining samples were imaged using a MZ16F stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik) or an SP2 confocal laser-scanning microscope (Leica). Images were processed using Fiji and Photoshop CS5 (Adobe) software. Brightness/contrast and color balance adjustments were always applied to the entire image. Quantifications were performed by hand using the “multi-point selection” tool of Fiji. Colocalization quantification was performed using the equivalent areas using the “ROI-manager” tool in Fiji. Nuclear area in Edu and IP experiments was measured using the “threshold” tool with the “moments” mask for all samples. Signal quantification of Yorkie antibody immunostaining was processed using Fiji software. Two planes were used to build the Z-projection. Nuclear-stained (DAPI) images were transformed into a mask using the “threshold” tool with the “moments” mask. The mask was used to obtain the nuclear signal with the Image calculator process. The nuclear signal obtained from the resulting image was measured to obtain the raw integrated density (RID). The nuclear area was obtained from the mask. Next, the RID was normalized to the respective nuclear area. Results were averaged per group and significant differences determined by 2-tailed Student t test.

In vivo images were obtained using Scmex 3.0 camera in a Zeiss Stemi SV 6 binocular loupe. WISH and whole-mount immunostaining images were captured with a ProgRes C3 camera from Jenoptik (Jena, TH, Germany). Cell counting of PH3+ staining was carried out by eye quantification in a previous defined area of each animal. Areas are schematically indicated in each figure. The total number of PH3+ cells was divided by the animal area. Double FISH confocal images were obtained with a Leica TCS SPE confocal microscope (Leica Microsystems, Mannhiem, BW, Germany). Representative confocal stacks for each experimental condition are shown. Images were blind analyzed and later grouped according to each genotype.

To prevent cell adhesion, p96-well plates were treated with 12 g/L poly-2-hydroxyethyl methacrylate (polyHEMA, Santa Cruz Biotechnology) resuspended in ethanol. Once the plates were dry, the cells were seeded at density of 1 cell per well. Spheroids were growth for 18–21 days in DMEM/F12 (Gibco) medium supplemented with 1% B27 (Gibco), 0.02 µg/mL EGF (Gibco), 0.004 µg/mL bFGF (ThermoFisher), 8% BSA (Nzytech), and 1% penicillin/streptomycin. Images were taken using a Nickon Eclipse Ti-S microscope and a JenoPTIK ProgRes C3 camera. Spheroid volume was estimated using the formula: volume = (length x width)² × 0.526.