Low Osteogenic Yield in Human Pluripotent Stem Cells Associates with Differential Neural Crest Promoter Methylation

Human pluripotent stem cell‐derived osteoblasts possess great potential for use in bone disorder elucidation and repair; however, while the general ability of human pluripotent stem cells to differentiate into osteoblasts and lay down bone‐specific matrix has been shown, previous studies lack the complete characterization of the process whereby such osteoblasts are derived as well as a comparison between the osteogenic efficiency of multiple cell lines. Here, we compared the osteogenic potential of two human induced pluripotent stem cell lines (RIV9 and RIV4) to human H9 embryonic stem cells. Generally capable of osteogenic differentiation, the overall osteogenic yield was lower in the RIV9 and RIV4 lines and correlated with differential expression of osteocalcin (OCN) in mature cultures and PAX7 and TWIST1 during early differentiation. In the undifferentiated cells, the promoters of the latter two genes were differentially methylated potentially explaining the variation in differentiation efficiency. Furthermore, the expression signatures of selected neural crest and mesodermal genes and proteins suggested that H9 cells preferentially gave rise to neural crest‐derived osteoblasts, whereas the osteoblasts in the RIV9 cultures were generated both through a mesodermal and a neural crest route although each at a lower rate. These data suggest that epigenetic dissimilarities between multiple PSC lines may lead to differences in lineage derivation and mineralization. Since osteoblast progenitors from one origin inadequately repair a defect in the other, these data underscore the importance of screening human pluripotent stem cells lines for the identity of the osteoprogenitors they lay down. Stem Cells 2018;36:349–362 The methylation status of the neural crest‐associated TWIST1 and PAX7 promoters in undifferentiated human pluripotent stem cells predicts whether they will differentiate into neural‐crest or mesoderm‐derived osteoblasts.