Method for differentiating mesenchymal stem cells into ligament-like tissue v1
Authors: Tsuru Michiyo 
Published: October 2017

Method for use with PuraMatrix peptide hydrogel The casting frame was placed at room temperature in a 1× PBS bath to initiate gelation. After 5 min, the gel slab was transferred to a Petri dish containing 4°C medium and subsequently placed in a 37°C incubator. Peptide gels were cultured in 10% FBS-supplemented feed medium. The slabs were seeded at a cell density of 15 × 106 cells/mL. Each slab was fed 12 mL of medium every other day. A peptide solution was added to a final peptide concentration of 0.5% and a cell density of 15 × 106 cells/mL. We washed the cells and peptide gel with a saccharose solution. At this time, sucrose solution was used to maintain physiological osmotic pressure. The culture medium was removed from stem cell-derived tissues cultured in a glass-bottom dish. The cells were washed twice with cold PBS and fixed in 4% paraformaldehyde in PBS. Rabbit PRELP antibody was used as the primary antibody and Alexa Fluor 488 goat anti-rabbit IgG was used as the secondary antibody. Nuclei were stained with DAPI and the actin cytoskeleton was visualized with Alexa Fluor phalloidin. The tissues were deparaffinized, washed twice in cold PBS, and fixed in 4% paraformaldehyde in PBS. A rabbit anti-PRELP (Sigma Aldrich) antibody was used as the primary antibody, and Alexa Fluor 488 goat anti-rabbit IgG was used as the secondary antibody. DAPI was used to stain the nuclei. After staining, the tissues were observed under CSLM (Olympus FluoView FV 1000-D) (Fig. 1-C). A ligament-like connective tissue composed of parallel fibers is observed (Fig. 1-D). Images of the ligament-like tissue that was cultured in a 3D gel are shown (20× objective). The tissue was exfoliated with a spatula, and the cells floated up from the bottom of the culture dish, which permitted us to observe their fibrous morphology. In this state, we were able to hold the tissue using dressing forceps. Cell counts were performed with the automated cell counter TC10 (BIO RAD, Inc). After 12 h, a BioStation ID cell analyzer (GE Healthcare) indicated the cells had transformed into a ligament fiber-like structure (Video 1). A three-dimensional culture showed that histogenesis was induced by introduction of the PRELP gene. Figure 1F shows the tissue at 7 days. The fibrous morphology was observed by causing the cells to float from the bottom of the culture dish, as followed by confocal scanning-laser microscopy (CSLM) with the laser on the Z-axis. The stem cells in the upper layer of the ligament-like tissue were 15 μm thick. Figure 1G shows the stem cell-derived ligament-like tissues cut into 3-μm slices and labeled with rabbit anti-PRELP (Sigma Aldrich) and Alexa Fluor 488 goat anti-rabbit IgG antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and visualized by CSLM. The ligament-like fibrous tissue was fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) and observed by transmission electron microscopy. Methods for use without PuraMatrix peptide hydrogel Transfected cells were cultured in 30 μM ZnCl2 and 10 ng/mL TNF-α to stem cells carrying the PRELP gene caused ligament tissue to appear after 24 h.