Safranin o

We induced in vitro differentiation into adipocytes, chondrocytes, and osteocytes as previously reported (Lee et al., 2010 (link)). Differentiated cells were stained by Toluidine blue (Wako Pure Chemical Industries), Safranin-O (Wako Pure Chemical Industries), and Alizarin red (Wako Pure Chemical Industries). We also differentiated them into myofibroblast (SMA+) and peripheral neurons (peripherin+) and performed an immunocytochemical analysis. In vivo differentiation of iPSC-NCCs into chondrocytes: We dissociated Method B iPSC-NCCs at day 10 with Accutase and purified the TRA-1–60-negative fraction using a MACS system (Miltenyi Biotec). We next injected 1.0 × 10⁶ TRA-1–60-negative cells into the testes of 8-week-old NOD-SCID mice, as previously described (Ohta et al., 2011 (link)). Eight weeks after transplantation, the testes were dissected and fixed with 4% PFA in PBS. The paraffin-embedded tissue was sectioned and stained with Toluidine blue (performed by Dept. of Pathology, Keio University School of Medicine). Images were obtained using a BZ-9000 (Keyence, Osaka, Japan) microscope.

The following materials were commercially obtained from the sources indicated: Dexamethasone, p-nitrophenyl-β-d-2-acetoamide-2-deoxyglucopyranoside, compound 48/80, an anti-dinitrophenyl IgE antibody (clone SPE-7), N-succinyl-Ala-Ala-Pro-Phe-pNA, mitomycin C, substance P, and dinitrophenyl human serum albumin (DNP-HSA) from Sigma-Aldrich (St. Louis, MO, USA), Toluidine blue, Safranin-O, and Evans blue from Wako Pure Chemical Industries (Osaka, Japan), an anti-trinitrophenyl IgE antibody (clone IgE-3) from BD Biosciences (San Diego, CA, USA), trinitrophenyl bovine serum albumin (TNP-BSA) from LSL (Tokyo, Japan), H-d-Ile-Pro-Arg-pNA (S-2288) from Chromogenix (Milano, Italy), N-(4-Methoxyphenylazoformyl)-Phe-OH potassium salt (M-2245) from Bachem AG (Bubendorf, Switzerland), an anti-G_αi2 antibody from Santa Cruz Biotechnology (Dallas, TX, USA), an anti-pan-actin antibody (clone C4), an anti-G_αi1 antibody, an anti-G_αi3 antibody, and thapsigargin from Merck Millipore (Billerica, MA, USA), a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody and a HRP-conjugated anti-rabbit IgG antibody from Agilent Technologies (Santa Clara, CA, USA) and recombinant mouse IL-3 from R & D Systems (Minneapolis, MN, USA). All other chemicals were commercial products of reagent grade.