Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Basic fibroblast growth factor (bFGF) regulates maintenance of stemness and modulation of osteo/odontogenic differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHEDs). Mineralization in the bones and teeth is in part controlled by pericellular levels of inorganic phosphate (Pi), a component of hydroxyapatite, and inorganic pyrophosphate (PPi), an inhibitor of mineralization. The progressive ankylosis protein (gene ANKH; protein ANKH) and ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1/ENPP1) increase PPi and inhibit mineralization, while tissue-nonspecific alkaline phosphatase (ALPL; TNAP) is a critical pro-mineralization enzyme that hydrolyzes PPi. We hypothesized that regulation by bFGF of mineralization in SHEDs occurs by modulation of Pi/PPi-associated genes. Cells were isolated from human exfoliated deciduous teeth and characterized for mesenchymal stem cell characteristics. Cells were treated with bFGF, and the osteogenic differentiation ability was determined. The mRNA expression was evaluated using real-time polymerase chain reaction. The mineralization was examined using alizarin red S staining. Cells isolated from primary teeth expressed mesenchymal stem cell markers, CD44, CD90, and CD105, and were able to differentiate into osteo/odontogenic and adipogenic lineages. Addition of 10 ng/ml bFGF to SHEDs during in vitro osteo/odontogenic differentiation decreased ALPL mRNA expression and ALP enzyme activity, increased ANKH mRNA, and decreased both Pi/PPi ratio and mineral deposition. Effects of bFGF on ALPL and ANKH expression were detected within 24 h. Addition of 20 mM fibroblast growth factor receptor (FGFR) inhibitor SU5402 revealed the necessity of FGFR-mediated signaling, and inclusion of 1 μg/ml cyclohexamide (CHX) implicated the necessity of protein synthesis for effects on ALPL and ANKH. Addition of exogenous 10 μm PPi inhibited mineralization and increased ANKH, collagen type 1a1 (COL1A1), and osteopontin (SPP1) mRNA, while addition of exogenous Pi increased mineralization and osterix (OSX), ANKH, SPP1, and dentin matrix protein 1 (DMP1) mRNA. The effects of PPi and Pi on mineralization could be replicated by short-term 3- and 7-day treatments, suggesting signaling effects in addition to physicochemical regulation of mineral deposition. This study reveals for the first time the effects of bFGF on Pi/PPi regulators in SHEDs and implicates these factors in how bFGF directs osteo/odontogenic differentiation and mineralization by these cells.