Differentiation of Model Loeys-Dietz Syndrome Type-II MiPSCs Into Ameloblast-Like Cells

Objectives: Loeys-Dietz Syndrome (LDS) is a connective tissue disorder caused by mutations along the TGF-Beta signaling pathway and characterized by cardiovascular, craniofacial, and oral/dental anomalies. Patients with mutations in TGFBR2 (LDS2) have severe enamel defects which alter enamel rod decussation with no significant changes in enamel volume or mineral density. Our study aims to differentiate iPSCs derived from a mouse model of LDS2 (Tgfbr2^G357W/+) into ameloblast-like cells to further investigate the underlying molecular mechanism responsible for the enamel defect unique to LDS2.
Methods: Murine iPSCs generated from wild type (Tgfbr2^+/+) and LDS2 (Tgfbr2^G357W/+) mouse embryonic fibroblasts were differentiated into ameloblast-like cells. iPSCs were first cultured on feeder cells, expanded through feeder free culturing, then transferred to low-attachment dishes until embryoid bodies (EBs) formed. Next, EBs were plated onto fibronectin-coated dishes and differentiated into the ectodermal stage, followed by two dental epithelial cell differentiation steps (DEC1/DEC2). Samples were analyzed at each stage for expression of ameloblast markers using RT-qPCR, western blot, and immunofluorescent staining.
Results: Gene expression levels for ameloblast differentiation markers (Amelx, Ambn) were similar in both Tgfbr2^+/+ and Tgfbr2^G357W/+ with progressive increase in expression at DEC1 and DEC2. These findings were confirmed at the protein level, indicating the LDS2 mutation does not impact ameloblast differentiation capability. Additionally, in vivo observations have shown that the mutation in Tgfbr2 does not affect enamel volume and mineral density, but rather alters ameloblast cytoskeletal dynamics. This is under further investigation in our in vitro model.
Conclusions: We successfully differentiated iPSCs from Tgfbr2^+/+ and Tgfbr2^G357W/+ mice into ameloblast-like cells. Our in vitro results corroborated our in vivo findings that Tgfbr2 heterozygous mutation does not impact the ability of ameloblasts to produce enamel. This in vitro model is invaluable for our ongoing investigation of the mechanism leading to the unique enamel phenotype of loss of decussation associated with LDS2.