Brachyury

Cells at ~80% confluence were fixed with 3.7% formaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin (BSA) in PBS. Cells were stained with primary antibodies Nanog (1:200; rabbit polyclonal, #PA1-097, Thermo Fisher Scientific), Oct4 (1:200; mouse monoclonal, #75463, Cell Signalling), Sox2 (1:500; rabbit monoclonal, #97959, Abcam), TRA1-60 (1:100; mouse monoclonal, #41-100, Life Technologies, Carlsbad, CA, USA), Brachyury (1:20; goat polyclonal, #AF2085, R&D System), Sox17 (1:20; goat polyclonal, #AF1924, R&D System), Nestin (1:1000; mouse monoclonal, #60091, Stem Cell Technologies), and Nrf2 (1:100; mouse monoclonal, #SC-365949, Santa Cruz) and incubated overnight at 4 °C in a blocking buffer. Secondary antibodies goat anti-mouse Alexa-Fluor-488, goat anti-rabbit Alexa Fluor-594, and donkey anti-goat Alexa-Fluor-594 (all from Life Technologies) were used for detection. DAPI (Carl Roth) was used for nuclei counterstain. Images were acquired with a Leica DMi8 inverted microscope. The filter cubes and software were provided by Leica.

For 2D cultures, the cells were seeded on coverslips and allowed to adhere overnight. 3D MSCs were embedded in OCT Tissue Tek and cryosectioned at 5–7 μm. Sections and coverslips were fixed in 4% paraformaldehyde for 10 minutes followed by overnight immunostaining for Ki67 (4 μg/ml, Abcam). HIF1α (5 μg/ml, Abcam), Brachyury (15 μg/ml, R and D Systems) or CXCR4 (10 μg/ml, Chemicon (Millipore)) according to the manufacturers’ instructions with a DAPI nuclear counterstain. Exposure to 200 μM cobalt chloride for 4 hours was used as a positive control for hypoxia (HIF1α). Samples were then incubated with fluorescently-conjugated secondary antibodies (Alexafluor 488 rabbit anti-mouse 1:500; Sigma rabbit anti-goat Cy3 1:400; Sigma goat anti-rabbit Cy3 1:400), before nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). Slides were imaged using the LSM510 confocal imaging system. LSM images of Brachyury immunostaining were analyzed using Volocity image analysis software to calculate % Brachyury-positive nuclei against DAPI staining. Volocity was also used to identify positions of the lowest and highest Brachyury immunofluorescent intensities in 48 of 761 cells each to determine distribution of positivity.

For the paraffin section, the Matrigel was broken with a cold KBM (Lonza). The hAOs were fixed in 4% PFA for 30 min and dehydrated with graded ethanol solutions for 30 min. Then, the blocks were embedded into paraffin and made into 4-μm sections. For hematoxylin and eosin (H&E) staining, the sections were rehydrated and then were stained. For immunofluorescence, antigen retrieval was achieved by citrate buffer, pH 6.0 at 121°C or 10 μg/mL proteinase K at 37°C. After cooling down, slides were treated with blocking solution, and the following first antibodies were used: OCT3/4 (Santa Cruz, TX, United States), OTX2 (R&D systems), Brachyury (R&D systems), SOX17 (R&D systems), PCNA (Abcam, CAM, United Kingdom), cleaved caspase-3 (Cell Signaling Technology, MA, United States), E-cad (BD biosciences, CA, United States), cytokeratin 14 (Abcam), p63 (GeneTex, CA, United States), collagen IV (Abcam), PITX2 (Abnova, Taiwan), DLX3 (Invitrogen, OR, United States), amelogenin (AMELX; Santa Cruz), and ameloblastin (AMBN; Biorbyt, UK, dentin sialoprotein (DSP; Santa Cruz), periostin (Abcam), cementum protein (CEMP1; Abcam), and human leukocyte antigen (hLA; Abcam). For visualization, anti-mouse or rabbit IgG conjugated with Alexa Fluor 488 or 555 dye (Invitrogen) was applied and observed under a confocal laser microscope (DMi8; Leica, Germany).

Human iPSCs (PKD KO and wildtype control) were differentiated into the 3 germ layers according to the STEMdiff Trilineage Differentiation Kit protocol (STEMCELL Technologies). Briefly, cells were seeded onto Matrigel-coated plates and treated with endoderm and mesoderm medium for 4 days and ectoderm medium for 6 days. Differentiated cells were then fixed and stained with antibodies against lineage-specific markers for ectoderm, endoderm and mesoderm as described above. Primary antibodies used were CXCR4 (R&D system mab173, 1:500), SOX17 (R&D system af1924, 1:20), Brachyury (R&D system af2085, 1:100), Nestin (R&D system mab1259, 1:500) and PAX6 (Stemgent 09-0075, 1:200).

Cells were fixed with 4% (vol/vol) paraformaldehyde (PFA) and subjected to immunostaining using the following primary antibodies: NANOG (1:1000; rabbit polyclonal, Abcam), OCT4 (1:400 mouse monoclonal, STEMCELL Technologies) for pluripotency, and BRACHYURY (1:20 goat polyclonal, R&D systems), SOX17 (1:20 goat polyclonal, R&D systems) and NESTIN (1:1000 mouse monoclonal, STEMCELL Technologies) for the three germ layers detection. After incubation with primary antibodies, cells were incubated with Alexa‐Fluor‐647, ‐594 and ‐488 conjugated secondary antibodies (all from Thermo Fisher Scientific) for 1 hour at 37°C. Nuclei were counterstained using 1 µg/mL Hoechst 33528 (Thermo Fischer Scientific). Microscopy was performed using imaging systems (DMi8), filter cubes and software from Leica microsystems. AP staining was performed using the 1‐Step NBT/BCIP (Thermo Fisher Scientific). Fluorescence quantization was achieved by measurement of the corrected total cell fluorescence (CTCF = Integrated Density – [Area of selected cell × Mean fluorescence of background readings]). Five cells/condition were randomly selected for analysis.

iPSCs were plated onto Geltrex™-coated glass coverslips. Directed in-vitro differentiation to the three germ layers was performed using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN, USA; SC027B) according to the manufacturer’s instructions. Primary antibodies used provided with the kit were: SOX17 (1:1000, R&D Systems, Minneapolis, MN, USA; #963121), OTX2 (1:1000, R&D Systems, Minneapolis, MN, USA; #963273) and BRACHYURY (1:1000, R&D Systems, Minneapolis, MN, USA; #963427). The secondary antibody used was Alexa Fluor 647 Donkey anti-Goat IgG (H+L; 1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; A21447). Cells were fixed, stained and imaged as previously described [27 (link)].