Hyrax c20

STEMs produced using fluorescent protein expressing cells were harvested on day 15 by placing a few drops of PBS through the wells, fixed with 3.7 % formaldehyde and then embedded in OCT (VWR, Germany) overnight. The STEM spheroids were then sectioned into 10 μm sections using a cryo-stat (HYRAX C20, Zeiss), transferred onto slides (Superfrost, VWR, Germany), stained with DAPI nuclear stain, and then imaged using a Zeiss Cell Observer Z1 (Carl Zeiss, Germany) fluorescent microscope. Imaging of spheroids after live/dead staining images were acquired using a Zeiss LSM 510 confocal miscrocope.

For histological staining, spheroids were collected and washed in DPBS, and fixed in 3.7% formaldehyde DPBS overnight, and then transferred to 30% sucrose for embedding into OCT medium for cryosectioning. Slices (6 µm thickness) were obtained using a Hyrax C20 (Carl Zeiss, Oberkochen, Germany) set at −26 °C. The staining was done according to the following immunohistochemistry procedure: Sections were washed in PBS and blocked with 2.5% goat serum, 0.1% Triton-X, 0.05% Tween20 in PBS, and then the samples were incubated with the primary antibody (Fibronectin, 1:300, Abcam; CD31, 1:100, Abcam; CD105 1:100, Santa Cruz Biotechnology; Pan-keratin, 1:100, Abcam) overnight and washed afterwards before counterstaining with hematoxylin. Control sections were processed without the primary antibody. Cultures grown in 2D were directly stained without the need for sectioning.
For live-dead staining (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, Waltham, MA, USA), spheroids were collected and washed twice with DPBS. DPBS was aspirated and cells were stained with 2 µM calcein AM (live) and 5 µM ethidium homodimer-1 (EthD-1, dead) DPBS solution for 30–60 min. After the incubation, cells were washed with DPBS once and imaged.