ACVR1R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

Macrophages play crucial roles in many human disease processes. However, obtaining large numbers of primary cells for study is often difficult. We describe 2D and 3D methods for directing human induced pluripotent stem cells (hiPSCs) into macrophages (iMACs). iMACs generated in 2D culture showed functional similarities to human primary monocyte-derived M2-like macrophages, and could be successfully polarized into a M1-like phenotype. Both M1- and M2-like iMACs showed phagocytic activity and reactivity to endogenous or exogenous stimuli. In contrast, iMACs generated by a 3D culture system showed mixed M1- and M2-like functional characteristics. 2D-iMACs from patients with fibrodysplasia ossificans progressiva (FOP), an inherited disease with progressive heterotopic ossification driven by inflammation, showed prolonged inflammatory cytokine production and higher Activin A production after M1-like polarization, resulting in dampened responses to additional LPS stimulation. These results demonstrate a simple and robust way of creating hiPSC-derived M1- and M2-like macrophage lineages, while identifying macrophages as a source of Activin A that may drive heterotopic ossification in FOP.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-ß) signaling is associated with osteoarthritis (OA). TGF-ß is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-ß's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-ß signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-ß that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-ß regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-ß type I receptor inhibitor, SB431542, was used to attenuate TGF-ß signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-ß receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-ß signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-ß signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.